Forensic DNA Analysis: Methods and Protocols (Methods in Molecular Biology, 2685) [1st ed. 2023] 1071632949, 9781071632949

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Methods in Molecular Biology 2685

Catherine Cupples Connon  Editor

Forensic DNA Analysis Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Forensic DNA Analysis Methods and Protocols

Edited by

Catherine Cupples Connon Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA

Editor Catherine Cupples Connon Department of Forensic Science Virginia Commonwealth University Richmond, VA, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3294-9 ISBN 978-1-0716-3295-6 (eBook) https://doi.org/10.1007/978-1-0716-3295-6 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface This volume of the well-known Methods in Molecular Biology series will focus exclusively on methods specific to forensic DNA analysis. Included in this series is a comprehensive collection of extraction, quantification, STR amplification, and detection methods for routine forensic samples, including a variety of manual, semi-automated, and automated procedures using both home-brew and commercial products. Also included are protocols for a probabilistic modeling software and specialized start-to-finish procedures for mitochondrial DNA analysis, archived latent fingerprints, latent DNA, rapid DNA profiling, and next-generation sequencing. This is truly a one-of-a-kind compilation of forensic DNA analysis procedures that will be the definitive laboratory protocol resource for all forensic DNA laboratories. Richmond, VA, USA

Catherine Cupples Connon

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Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

INTRODUCTION

1 Forensic DNA Analysis: An Overview of the Laboratory Process. . . . . . . . . . . . . . Catherine Cupples Connon

PART II

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3

DNA EXTRACTION AND PURIFICATION

2 Organic Extraction of Nucleic Acids Using Ethanol Precipitation or Microcon® Centrifugal Filter Purification Methods . . . . . . . . . . . . . . . . . . . . . . . 23 Carolyn A. Lewis 3 Manual Silica-Based DNA Extractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Catherine Cupples Connon 4 Applied Biosystems™ PrepFiler™ Forensic DNA Extraction Kit (Manual and Semi-automated via AutoMate Express™). . . . . . . . . . . . . . . . . . . 53 Megan M. Foley 5 Robotic DNA Extraction Utilizing Qiagen BioSprint® 96 Workstation . . . . . . . . 83 Brittany Ziencik 6 DNA Extraction of Bone Through Demineralization . . . . . . . . . . . . . . . . . . . . . . . . 93 Brandi L. Iorio and Ashley M. Cooley 7 Differential Extraction with Purification via Organic/Microcon® and Promega DNA IQ™ Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Jonathan Forsberg and Caitlin Ayoub 8 DNA Purification from Bloodstains and Buccal Cells/Saliva on FTA® Cards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Brittany C. Hudson and Catherine Cupples Connon

PART III

DNA QUANTIFICATION

9 Yield Gel via Quantitative Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Victoria R. Parks and Dayanara A. Torres 10 Quantitative PCR of Alu Repeats Using PowerUp™ SYBR® Green Master Mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sierra L. Laveroni and Victoria R. Parks 11 Quantitation of DNA Using the Applied Biosystems Quantifiler® Trio DNA Quantification Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kelly L. Knight, Angelina Mauriello, and Georgia Williams 12 QIAGEN’s Investigator® Quantiplex® Pro Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michelle D. Bonnette

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Contents

PART IV 13

14

15 16 17

DNA Amplification Using Promega’s PowerPlex® Fusion Systems (5C and 6C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Caitlin McCaughan and Kristy A. Lenz Amplification of Extracted DNA and Direct Amplification with the PowerPlex® Y23 System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jonelle M. Thompson Applied Biosystems’ GlobalFiler™ PCR Amplification Kit . . . . . . . . . . . . . . . . . . . Georgia Williams, Megan M. Foley, and Kelly L. Knight QIAGEN’s Investigator® 24plex QS and GO! PCR Amplification . . . . . . . . . . . . Michelle D. Bonnette Low Volume STR Amplification Options: Coupling with Standard or Fast PCR, Traditional or Normalized DNA Extraction, and/or Traditional or Alternative Capillary Electrophoresis . . . . . . . . . . . . . . . . . . Catherine Cupples Connon

PART V 18

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STR AMPLIFICATION 207

227 241 253

263

STR PROFILE DETECTION AND INTERPRETATION

Capillary Electrophoresis with Applied Biosystems’ 3500 Genetic Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 Kara Kovach Likelihood Ratio Calculation Using LRmix Studio . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Megan M. Foley

PART VI

SPECIALIZED SAMPLES

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Mitochondrial DNA Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ashley M. Cooley 21 An Optimized Forensic DNA Analysis Workflow for Obtaining STR Results from Archived Latent Fingerprints . . . . . . . . . . . . . . . . . . . . . . . . . . . . April D. Solomon 22 Detection of Latent DNA Using a DNA Binding Dye. . . . . . . . . . . . . . . . . . . . . . . Adrian Linacre and Piyamas Petcharoen 23 Rapid DNA Profile Development with Applied Biosystems RapidHIT™ ID System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Megan M. Foley 24 Next-Generation Sequencing: ForenSeq™ DNA Signature Prep Kit with the Illumina MiSeq FGx . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Megan M. Foley

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors CAITLIN AYOUB • Virginia Department of Forensic Science, Richmond, VA, USA MICHELLE D. BONNETTE • InVita Healthcare Technologies, Jacksonville Beach, FL, USA CATHERINE CUPPLES CONNON • Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA ASHLEY M. COOLEY • Virginia Department of Forensic Science, Richmond, VA, USA MEGAN M. FOLEY • Department of Forensic Sciences, The George Washington University, Washington, DC, USA JONATHAN FORSBERG • Virginia Department of Forensic Science, Richmond, VA, USA BRITTANY C. HUDSON • Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA; Integrative Life Sciences, Virginia Commonwealth University, Richmond, VA, USA BRANDI L. IORIO • Virginia Department of Forensic Science, Richmond, VA, USA KELLY L. KNIGHT • Forensic Science Program, George Mason University, Fairfax, VA, USA KARA KOVACH • Erie County Central Police Services Forensic Laboratory, Buffalo, NY, USA SIERRA L. LAVERONI • Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA KRISTY A. LENZ • Promega Corporation, Madison, WI, USA CAROLYN A. LEWIS • Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA; Integrative Life Sciences, Virginia Commonwealth University, Richmond, VA, USA ADRIAN LINACRE • Forensic DNA Technology, College of Science and Engineering, Flinders University, Adelaide, SA, Australia ANGELINA MAURIELLO • Forensic Science Program, George Mason University, Fairfax, VA, USA CAITLIN MCCAUGHAN • Bexar County Criminal Investigation Lab, San Antonio, TX, USA VICTORIA R. PARKS • Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA PIYAMAS PETCHAROEN • Forensic Technology and Innovation Module, School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand APRIL D. SOLOMON • Jefferson Parish Sheriff’s Office Regional DNA Laboratory, Harvey, LA, USA JONELLE M. THOMPSON • Promega Corporation, Madison, WI, USA DAYANARA A. TORRES • Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA GEORGIA WILLIAMS • Forensic Science Program, George Mason University, Fairfax, VA, USA BRITTANY ZIENCIK • Virginia Department of Forensic Science, Richmond, VA, USA

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Part I Introduction

Chapter 1 Forensic DNA Analysis: An Overview of the Laboratory Process Catherine Cupples Connon Abstract Developing a suitable DNA profile from forensic evidence has long been a lengthy, multi-step laboratory process. Over the last couple of decades, the “process” has exploded into a plethora of numerous options for each of the individual steps, including different manufacturers and commercial kits, as well as options for manual, semi-automated, and automated processing. Despite these options, the heart of the big picture process remains fairly consistent with its early 2000s counterpart and is deeply embedded with a wide variety of precautions to help prevent contamination and ensure integrous results. This includes habitual cleaning, wearing personal protective equipment (PPE), using sterile products and reagents, processing controls, and employing strategic laboratory practices. This chapter serves to briefly introduce new audiences to the forensic DNA process, particularly from a laboratory perspective. Invaluable information regarding routine precautions is included here, and it is highly recommended that this chapter be read first, as much of the information applies to nearly all the chapters of this text. Key words Forensic DNA, Quality assurance, Quality control, Personal protective equipment, Precautions, Controls, Contamination

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Introduction The typical, modern laboratory process used to develop a DNA profile for human identification purposes from forensic evidence takes about a day or two—a vast improvement from decades earlier. Following an initial screening process, items that are deemed likely to yield a probative DNA profile are continued on to DNA extraction and purification, quantification, amplification of short tandem repeat (STR) loci, and profile detection via capillary electrophoresis. The resulting DNA—or more specifically, STR—profiles are then analyzed for accuracy, followed by comparison to profiles of other evidentiary items to form conclusions about the origins of DNA located on such items. Given that items of this nature tend to be highly compromised (e.g., little and/or low-quality DNA) and we are dealing with an alleged criminal act, it is of the utmost

Catherine Cupples Connon (ed.), Forensic DNA Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2685, https://doi.org/10.1007/978-1-0716-3295-6_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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importance for the forensic scientist to do everything possible to ensure the integrity of the evidence. This includes handling it with care so as not to waste, lose, contaminate, degrade, or otherwise further compromise the precious DNA. Many of these precautions are common laboratory practices for similar fields (e.g., a clinical setting), and their importance is made extremely clear by the fact that the forensic DNA community has strict, thorough quality assurance standards [1–3]. Furthermore, as technology has advanced, a variety of procedural options have come to the forefront, and laboratories have the benefit of piecing together the extraction, quantitation, amplification, detection, and even analysis software methods of their choosing. This includes not only a variety of manual, semi-automated, and automated procedures, but also the selection of commercial products and instruments from a variety of well-established manufacturers, such as Applied Biosystems, Promega, and Qiagen.

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Universal Precautions Against Contamination and Compromise Technological advancements have made the DNA profiling process extremely sensitive, furthering the need to take extreme precautions against contamination. General “universal precautions” are taken such that the analyst should assume that they will (inadvertently) contaminate the evidence item if they are not immensely careful and that the evidence itself is highly infectious with a lifethreatening agent. Yes, all very extreme scenarios; however, they are intended to make the analyst recognize not only how important these precautions are but also to strictly adhere to them. There is a laundry list of precautions that we always adhere to as forensic DNA analysts: restrict access to work areas to authorized personnel only; physically partition work areas based on tasks performed (e.g., pre-amplification and post-amplification); wear PPE; clean our workspace, equipment, and instrumentation before and after use; use sterile plastics, reagents, etc.; handle one sample at a time; and utilize controls. It is the laboratory’s responsibility to not only precisely define how these precautions will be employed but also ensure that analysts are trained and monitored appropriately. Laboratory space needs to be secure and restricted to laboratory staff and other authorized users. The more human traffic there is in such spaces, the more likely someone will leave their DNA behind (see Note 1). Physical separation of work areas may also be necessary—or at least beneficial—depending on what procedures are being performed. Forensic standards require physical separation (i.e., different rooms) of routine DNA casework from DNA databasing, as well as separation of rapid DNA profiling from both of these other testing laboratory spaces [1, 2]. Additionally, pre-amplification procedures (e.g., sample accessioning, screening,

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extraction, polymerase chain reaction (PCR) setup, etc.) must be conducted at different times or in separate spaces from one another, while the generation and further processing of PCR amplified product must reside in a completely separate room(s) from pre-amplification activities [1, 2]. These are routinely referred to as “pre-amplification” and “post-amplification” laboratory spaces. Utilizing separate laboratories for different profiling techniques— such as STR, mitochondrial DNA, and next-generation sequencing (NGS)—is also highly recommended. When in the laboratory, individuals should always wear PPE, including but not limited to lab coat and gloves. No one should ever touch anything in the laboratory without gloves, as they can leave trace amounts of their genetic material behind, which could later be transferred to someone else’s gloves if they happen to come in contact with the same surface. Once genetic material is on their gloves, there is a risk that it could then be transferred to an item of evidence, a reagent, etc., and contaminate the resulting DNA profile. Even when wearing gloves, analysts are not completely safeguarded and need to be mindful of what they touch. They should not touch their face, hair, clothing/shoes, etc. They should be careful about not touching doorknobs, telephones, personal items, chair backs, etc. For any of these “touching” events, their gloves must be changed immediately. Additionally, hair must be secured, and, in some settings (e.g., mitochondrial DNA analysis), hair nets must be worn. Legs and feet must be appropriately covered by pants and shoes (e.g., closed-toe shoes that also cover the top of the foot, no shorts, etc.). Many laboratories will find it advantageous to require face coverings/masks, as talking over and/or near evidence without a face covering can result in inadvertent transfer of DNA from small droplets/aerosols of saliva. On a related note, excessive talking in the laboratory can be distracting and result in an analyst(s) making some kind of procedural error. Additionally, sleeve guards and/or shoe coverings may need to be worn. The latter two items are not as often encountered in a typical forensic DNA laboratory but may be necessary depending on the laboratory itself. Safety glasses are also a common form of PPE, but when employed in a forensic DNA setting, they are usually worn to protect the wearer from harmful chemicals, infectious agents, etc., rather than to protect the evidence from contamination. DNA laboratories should also have a clear policy regarding a cleaning/decontamination routine. These can be broken up into different types of cleaning associated with performing a procedure versus periodic cleaning (e.g., daily, weekly, monthly, quarterly, etc.) that is scheduled regardless of whether a laboratory procedure is going to be/was performed on a given day. If a procedure is to be performed, the surfaces, equipment (including pipettes, tip boxes, forceps/tweezers, scissors, etc.), and instruments (e.g., doors, keypads/user interface screens/touchpads, etc.) should be

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decontaminated by thoroughly wiping down with 10% bleach, followed by 70% ethanol, using a fresh laboratory paper towel for each. Bench pads (also known as lab soak, lab diapers, etc.) should be used as a disposable workspace for the procedure, rather than working directly on the bench itself. These should be changed on an as-needed basis during the procedure. At the end of the procedure, the bench pad should be discarded, and the bench top, equipment, and instruments disinfected again with bleach and ethanol. Equipment (e.g., pipettes, forceps, and scissors) can be exposed to ultraviolet (UV) light as an additional decontamination measure; autoclaving is also a decontamination option for some items (e.g., forceps and scissors). Plastics, reagents, and other consumables used for forensic DNA laboratory testing must be sterile—or more precisely, free of DNA/genetic material—before beginning any procedure. Just because pipette tips, microcentrifuge tubes, etc., come in a secured container from the vendor, they are not necessarily free of extraneous DNA. They may have been inappropriately handled at any one (or more) of numerous stages prior to use in the forensic laboratory. The most responsible action is to autoclave these plastics prior to use and limit handling after that. Never handle these items without gloves, and never reach into a container of autoclaved tubes, even when wearing gloves; instead, gently pour out the number of tubes needed onto a clean lab wipe, cap them, and arrange them in your tube rack. Never handle a pipette tip directly; insert the end of the pipette shaft into the opening of the tip while it is still racked in the tip box and tap down to secure it on to the end of the pipette. Use of aerosol-barrier pipette tips are also recommended because they protect against small particles (like dust and aerosols) from falling into the tips and ending up in your reagent/ sample, as well as to protect the end of the pipette shaft itself from coming into contact with a reagent/sample due to accidental overpipetting, bubbles, etc. You should likewise be confident that the reagents you are using are sterile. These can be purchased sterile from the vendor or autoclaved after recipient/in-house preparation (if acceptable for that reagent/container; see Subheading 4.1). Prior to the use of a critical reagent (see Note 2) with a lot number that has not been used before, it must be tested to ensure it is not contaminated and yields the expected results; this is part of the quality control process [1–3]. It is best practice to make a small aliquot of a reagent (e.g., ~1–50 mL) for your own personal use rather than pipetting from the larger stock reagent. This reduces the risk of contaminating the larger stock reagent; if your small aliquot becomes contaminated, the contamination is isolated to that small container and your samples alone. It is good practice to discard personal aliquots after about a month, or sooner, if needed. Other consumables, such as cotton swabs for sample collection, should be sterilized prior to use; it is best to purchase these sterile,

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rather than attempting to sterilize them in-house. The bottom line with respect to these consumables is that if you are ever in doubt, throw it out! It is not worth compromising one or more of your samples if you suspect you many have contaminated a tip, tube, reagent, etc. While working with samples, handle only one sample at a time. If working with the actual item of evidence, the gloves, scissors, forceps, bench pad, etc., should be changed/decontaminated between each item, and the item should be securely returned to its packaging before proceeding to another item. As cuttings of these samples are transferred to individual microcentrifuge tubes for DNA testing, those tubes should be labeled appropriately so that they can be identified at any given time. Laboratory-approved worksheets should be prepared for these samples prior to starting a multi-sample procedure to help guide you along the way and make sure all samples are processed; these worksheets also help document in what order samples are processed, as well as where they are located in a multi-sample plate (e.g., a 96-well plate) or on an instrument. Individual sample tubes should be checked as you progress through the procedure to ensure you are always working with the correct sample. It is a helpful habit to physically move a sample tube to a different column/row/location of the tube rack after you have completed a step so that you can keep track of where you are in the process at any given time. All of these measures help prevent sample switches. Handle and pipette into/out of the sample tubes with care. This includes opening each tube slowly and carefully to prevent small droplets of liquids (aerosols) from spraying out into the air or onto your gloves. If this occurs, immediately decontaminate the affected surface(s) and/or change your gloves. Only one tube should be open at a time; when opening/closing it, be careful not to touch the inside of the cap with your glove. If that happens, change your glove(s) immediately. While the tube is open, be careful not to spill its contents (immediately clean up, change bench pad, etc., if this happens) or allow unintended particles to enter the tube. Working in a chemical fume hood or biosafety cabinet can help prevent the latter. Never reach over an open tube, uncovered/exposed sample plate, open box of tips, etc., as particles from your lab coat could fall into any of these containers or you could knock the container over and spill its contents. When pipetting to/from a tube (again, with only one tube open at a time), pipette slow and steady; be mindful of the first and second stops of the pipette plunger. Prior to pipetting, it is common practice to allow the sample/reagent you are pipetting from to come to room temperature (if previously stored at
20  C, 4  C, etc.), followed by vortexing and a quick spin. After aspirating (drawing up liquid into the tip) from that tube, check the volume of the liquid in the pipette tip to make sure it looks about right for

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the intended volume, that there are no air bubbles present, etc. After dispensing the reagent/sample to its intended location, check the pipette tip again to make sure all was dispensed; pipette just past the first stop if needed, but be careful not to introduce bubbles. At this point, it is often appropriate to vortex and quick spin the tube in which sample/reagent was added (but follow the protocol, as sometimes this is not the case). Change pipette tips between each sample/reagent (to help prevent sample-to-sample contamination), and always keep the tip box closed when not getting a tip (to help prevent contamination of the tips). If your pipette tip ever accidently touches something it shouldn’t (e.g., the bench pad, counter, another tube, your lab coat/glove/hand, etc.), change it immediately. The final general precaution that we utilize in forensic DNA testing is the use of controls. Controls are of known origin and serve two basic purposes: to ensure that no reagent, other consumable, piece of equipment, etc., used in the procedure is contaminated and that the procedure worked as expected. The former are generally categorized as negative controls, while the latter are categorized as positive controls. Specific controls are introduced at each step of the DNA analysis process, and many, but not all, are carried through to the final step.

3

Routine Guidance for DNA Processing

3.1 Separation of Question and Reference Samples

Forensic DNA samples can be placed into two broad categories: question/unknown and reference/known. As the names imply, some items are of unknown, or questioned, origin, like a red/brownish stain suspected to be blood that was collected from a knife or article of clothing, or a yellowish stain suspected to be a mixture of semen and vaginal fluid that was collected from the underwear of a sexual assault victim. On the other hand, some items are of known origin, as they are collected as reference samples from a person (hopefully of known identity) either to be used for comparison purposes in a specific case or to be entered into a DNA database; these are typically buccal swabs collected from the cheek area of the inside of the individual’s mouth or as venous blood collected from their arm. In forensic DNA casework, the DNA profiles obtained from the question samples are compared to those obtained from the reference samples in an attempt to determine the origin of the genetic material from the question samples. For forensic DNA databasing, the profiles will be stored in a restricted-access database for subsequent comparisons. Given the nature of these ultimate goals, it is customary that question samples are processed prior to and separate from reference samples. Additionally, question samples tend to be more compromised compared to the generally high-quality and high-quantity

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reference samples, the latter of which tend to yield high-quality DNA profiles without as much effort as compared to the lowerquality question samples. Thus, laboratory procedures for question samples tend to be designed and optimized for a particular sample type, whereas the procedures used for reference samples tend to work for most reference samples (see Note 3). 3.2 DNA Extraction and Purification

Following screening (see Note 4) and the selection of suitable/ promising DNA samples, samples should be grouped together by sample type (question separate from reference), and further sub-grouped by the extraction process to be utilized. This grouping is often referred to as a “batch” or a “run” and must contain its own set of extraction controls [1, 2]. The positive control is of known origin, and the negative control is a reagent blank; both are processed as if they were any other sample, except no DNA/substrate is added to the reagent blank. The positive control is not required by the forensic DNA quality assurance standards, but many laboratories still choose to process one (see Note 5), while the reagent blank must be carried through the entire process to capillary electrophoresis detection [1, 2]. A variety of manual and semi-automated extraction methods are available. Batch sizes are generally limited by the subsequent DNA quantification step (typically performed on a 96-well format), equipment (e.g., centrifuge space), and/or semi-automated instrument space (usually 6–16 samples for low-throughout options and 96 samples for high-throughput options). All of the routine laboratory techniques and universal precautions should be followed (see Subheading 2), especially having only one tube open at a time. The initial step of nearly all of the extraction procedures is a cell lysis step that is dependent on numerous reagents. Rather than adding each reagent one at a time to each tube, an extraction buffer master mix/cocktail of all of the reagents can be made. If utilizing this strategy, be sure to make enough for all samples in the batch (including controls and accounting for a small amount of extra volume needed due to pipetting errors) and mix/vortex thoroughly prior to dispensing to each sample. Moreover, this is a fairly high-risk part of the overall process with regards to contamination and sample switches. The lysis process utilizes a detergent, which tends to be bubbly and can make a mess when pipetting—potentially resulting in reagent coming out of the tube when it is capped—if not careful. There are many vortexing steps, so it is important to ensure that tubes are securely closed. Many of the extraction or purification protocols call for tube transfers, so it is imperative that samples are not only labeled correctly but also transferred correctly.

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3.3 DNA Quantification

Following extraction and purification, the amount of human DNA must be determined for question samples prior to continuing on to profile development [1]. Reference samples can also be quantified with a human-specific method, bypassing this process entirely, or use a non-human quantitation method (see Note 6); whichever option a laboratory chooses, they must validate it [1, 2]. At this stage, the controls initiated at the extraction process must also be processed, as well as DNA standards (or a calibrator, if using a virtual standard curve) [1, 2]. Laboratories can choose to initiate additional controls at this point, such as a calibrator (positive control) and/or no template control (NTC; negative control), which should be processed concurrently with the associated batch samples. A calibrator is of known origin and DNA concentration. An NTC is another type of reagent blank that consists of all of the reagents for the quantitation method but lacks DNA template; no water or buffer is added in its place. If either of these controls are employed at this step, they stop here; they are not processed beyond quantitation. A variety of commercial products are available for quantification of human DNA. Nearly all of these utilize real-time quantitative PCR (qPCR) with a 96-well format thermal cycler enhanced with an optical filter to detect fluorescence; this is often simply referred to as a real-time PCR instrument. These “quant” plates tend to be as close to 96 samples/controls as possible, if not entirely full, and can be setup manually or via a liquid handler instrument to pipette the reagents. This is often the result of combining smaller extraction batches together for a single “quant run.” As a costsavings measure, half reactions are often used in place of full reactions. All of the routine laboratory techniques and universal precautions should be followed (see Subheading 2), especially avoiding reaching over an open/exposed 96-well plate. Given the extremely sensitive nature of PCR, this process should be setup in the protection of a biological safety cabinet or hood. Additionally, since these batches tend to fill or nearly fill a 96-well plate, a PCR master mix is made that consists of all of the necessary components, except for the DNA template (extract). It is imperative that all reagents are brought to room temperature, vortexed, and quick-spun prior to making the master mix (see Note 7). The master mix should be enough for all samples/controls in the batch and have enough extra for pipetting error. Similar to the master mix reagents, the DNA extracts should have time to come to room temperature and should be vortexed and quick-spun prior to addition to the reaction plate. Sealing the plate securely with an adhesive film is essential to prevent evaporation. The plate should be spun in a plate spinner/ centrifuge prior to being loaded on the real-time instrument to remove bubbles that may have formed during setup. These last two

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precautions help to ensure that each well of the 96-well plate undergoes an efficient PCR reaction. 3.4 STR Amplification

Following quantitation, samples are normalized to a concentration that allows a specific nanogram amount (~0.25–1.0) of DNA to be PCR amplified in order to develop an STR profile that aids in human identification. Similar to the quantitation step, the reagent blank from the extraction step must be amplified under the same conditions as the DNA extracts (see Note 8), and a new set of controls are initiated at this point, which must be amplified at the same time as the associated batch samples [1, 2]. The positive amplification control is of known origin and DNA concentration. The negative amplification control is another type of reagent blank that consists of all of the reagents for the amplification method but lacks a DNA template; whatever is used to normalize/dilute the DNA extracts for this step (e.g., Type I water or TE
4) is added in place of DNA to the amplification negative control. Like the reagent blank from the extraction control, both of these amplification controls are carried through the entire process. Similar to extraction and quantitation, there are numerous commercial kits available for STR amplification. Most of the autosomal STR amplification kits that are currently available target all 20 of the CODIS loci, plus more. Like qPCR, these amplification batches are limited by the number of wells in the thermal cycler in which they will be amplified—typically 96 or 384 wells. As a costsavings measure, half reactions are often used in place of full reactions for question and/or reference samples. Due to the highquality nature of reference samples, reaction volumes as low as 3 μL have been successfully implemented (~1/8th reaction) [4, 5]. Less than half reactions are generally not suitable for question samples due to their compromised nature and potential to contain DNA from more than one contributor (i.e., a mixture); both of these lead to peak height imbalances, which are further exacerbated by low volume reactions, making them unreliable for such samples. All of the routine laboratory techniques and universal precautions should be followed (see Subheading 2), especially avoiding reaching over open tubes or an exposed 96-well plate. Many of the additional precautions specific to qPCR also apply here (see Subheading 3.3): setting up the reactions in a biological safety cabinet or hood; strategically preparing a master mix for all samples/controls; securely sealing the amplification plate or individual tubes; and spinning down the plate/tubes prior to loading on the thermal cycler.

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3.5 STR Profile Detection

4 4.1

Following STR amplification, the amplification (“amp”) product needs to be separated based on sized and detected so that the resulting STR profile can be further reviewed; this takes place via capillary electrophoresis. At this point, the extraction and amplification controls are processed along with the samples (see Note 9). The positive and negative amplification controls typically serve as the detection controls as well. The other specialized reagents for separation and detection are linked directly to the STR amplification method that was used; these are typically provided as part of the purchased amplification STR kit, including an allelic ladder and internal size standard, though sometimes the size standard is purchased separately. The most current amplification kits for human identification typically rely on the use of a 5- or 6-dye system. All of the routine laboratory techniques and universal precautions should be followed (see Subheading 2), especially avoiding reaching over an open/exposed 96-well plate. Many of the additional precautions specific to amplification also apply here (see Subheadings 3.3 and 3.4): preparing the amplification product for detection in a biological safety cabinet or hood; strategically preparing a master mix for all samples/controls; securely sealing the detection plate with a plate septa; and spinning down the plate prior to loading on the capillary electrophoresis instrument.

Additional Guidance Water

Water is necessary for a variety of laboratory procedures, including reagent preparation, sample storage and dilution, etc. Despite its apparent simplicity, it is imperative that the appropriate water is used for forensic DNA testing. Reagent water is classified based on its purity with respect to properties such as resistivity, conductivity, total organic carbon (TOC), and bacteria count [6]. The American Society for Testing and Materials (ASTM) takes most of these properties into consideration when classifying water as Type I, Type II, Type III, and Type IV, but classifies based on bacteria count separately using Type A, Type B, and Type C (see Table 1). Each type of water can be used for various laboratory applications; classifications of A, B, and C are only assessed on an as-needed basis. For forensic DNA analysis, Type A is always needed for any application in which the water is used in conjunction with sample collection or subsequent testing that leads to a genetic profile. The primary focus here will be in the discussion of Types I–IV. Of these four types of water, Type I is the most pure—virtually pure, in fact—with a resistivity of 18–18.3 MΩ-cm at 25  C, conductivity of and space. The underscore is also displayed in the RapidLINK™ Software. Letters always appear in capitalized format.

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9. If the swab is >3 inches, the handle needs to be snapped or cut with sterile scissors or a scalpel. 10. The chamber fills with liquid and the sample may not stay submerged. The sterile swab helps anchor it to the bottom to allow for optimal extraction. 11. If the screen shows the cartridge with a red “X”, the cartridge is either expired or has been inserted improperly. If the latter, remove the cartridge and reinsert. If expired, obtain a new cartridge. All components have an RFID tag that is read by the instrument that identifies the component, the lot number, and the expiration date. 12. If priming of the instrument occurs during this particular run, expect run times of ~110 min. This does not occur every run. 13. If the cartridge appears to be stuck, it may still be locked in the instrument. An admin or supervisor needs to perform a recovery function (see Subheading 3.4, step 1). 14. Studies have been performed that show reprocessing of the sample may still yield another profile [5, 6, 8]. Evaluate and validate if this procedure is to be used. 15. If the results screen does not appear, the cartridge may have been removed too early. Insert the cartridge back in the instrument and repeat the step (see Subheading 3.1, step 14). 16. If the “Export” button is not visible after the USB device has been inserted, check that the USB device has been properly inserted. Remove and reinsert if needed. Additionally, check the configuration for run deletion. The user is able to choose the option to delete the run once it has been transferred to the RapidLINK™ Software through the use of the system’s network, and it may no longer be available in the RapidHIT™ ID System. 17. Perform this step on a routine schedule—once a week, biweekly, or monthly based on the usage of the instrument. 18. Primary cartridge replacement can only be performed by users with admin or supervisor privileges. Primary cartridge maintenance may also need to be performed if there is an instrument or reagent error. 19. If the instrument is used for RapidINTEL™ purposes, use a GlobalFiler™ Express primary cartridge. 20. Do not remove the foam casing around the gel cartridge. This remains during insertion into the primary cartridge. 21. Once this is removed, the capillary is exposed. Handle with care! 22. If using a RapidINTEL™ Cartridge, run the GFE allelic ladder.

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23. If the screen shows the cartridge with a red “X”, the cartridge is either expired or has been inserted improperly. If the latter, remove the cartridge and reinsert. If expired, obtain a new cartridge. 24. A green check mark means the profile was generated with no quality flags and expected alleles were detected in the positive control; no alleles were detected in the negative control; or the allelic ladder profile was generated with no flags, all expected alleles were detected, and the ladder has been uploaded to the instrument’s library for allelic ladders. A red “X” means that the positive control profile was not as expected (e.g., no profile was generated, not all alleles were detected, or too many alleles were detected); alleles were detected in the negative control; or not all alleles were detected for the allelic ladder. The affected control(s) must be re-processed; if contamination in the negative control persists, contact Thermo Fisher. 25. Must be signed in as an administrator. 26. If multiple instruments within the laboratory are on the same network, this procedure adds this user to all instruments connected. 27. Settings should be validated and determined for each laboratory based on the usage of their instruments. 28. Only .txt, .csv, and .xml CODIS files can be uploaded. 29. Each allele is listed in a separate cell. Y-specific loci should be removed from the file. 30. To perform this action, the instrument must be active and the user needs the host name of the instrument. 31. If connected to the network, Google Maps can fill in this information. 32. The instrument must be connected to the network. If the user still has access, repeat this action after confirming connection. 33. These settings can be used for the GFE runs. The Intel and NGM runs require different settings based on validation. References 1. Federal Bureau of Investigation (2022) Rapid DNA. Available via Federal Bureau of Investigation. https://www.fbi.gov/services/labora tory/biometric-analysis/codis/rapid-dna. Accessed 21 Apr 2022 2. Hess AS (2015) FBI’s plans for the use of rapid DNA technology in CODIS. Available via Federal Bureau of Investigation. https://www.fbi. gov/news/testimony/fbis-plans-for-the-useof-rapid-dna-technology-in-codis. Accessed 21 Apr 2022

3. Kartasin´ska E, Jurga A (2020) Rapid DNA—a technology for rapid automated DNA profile analysis based on STR loci polymorphism. Issues Forensic Sci 309(3):33–40. https:// doi.org/10.34836/pk.2020.309.1 4. Federal Bureau of Investigation (2020) Quality Assurance Standards for forensic DNA testing laboratories. Available via Federal Bureau of Investigation. https://www.fbi.gov/file-reposi tory/quality-assurance-standards-for-forensic-

DNA Profile Development with RapidHIT™ ID System dna-testing-laboratories.pdf/view. Accessed 21 Apr 2022 5. Hennessy LK, Mehendale N, Chear K et al (2014) Developmental validation of the GlobalFiler® Express kit, a 24-marker STR assay, on the RapidHIT® System. Forensic Sci Int Genet 13:247–258. https://doi.org/10. 1016/j.fsigen.2014.08.011 6. Wiley R, Sage K, LaRue B et al (2017) Internal validation of the RapidHIT® ID System. Forensic Sci Int Genet 31:180–188. https:// doi.org/10.1016/j.fsigen.2017.09.011 7. Federal Bureau of Investigation (2022) Guide to all things rapid DNA, Version 1.0. Available via Federal Bureau of Investigation. http:// www.lsp.org/pdf/FBI_Guide_to_All_Things_ Rapid_DNA_01_27_2022.pdf. Accessed 21 Apr 2022 8. Salceda S, Barican A, Buscaino J et al (2017) Validation of a rapid DNA process with the RapidHIT® ID System using GlobalFiler® Express chemistry, a platform optimized for decentralized testing environments. Forensic Sci Int Genet 28:21–34. https://doi.org/10. 1016/j.fsigen.2017.01.005 9. Applied Biosystems (2020) APPLICATION NOTE—RapidHIT ID System: bone sample processing on the RapidHIT ID System with RapidINTEL cartridges. Available via Thermo Fisher Scientific. https://assets.thermofisher. com/TFS-Assets/GSD/Application-Notes/ Bone-sample-processing-RapidHIT-ID-sys tem-RapidINTEL-cartridges-applicationnote.pdf. Accessed 21 Apr 2022 10. Watherston J, Watson J, Bruce D et al (2022) An in-field evaluation of rapid DNA instruments for disaster victim identification. Int J Legal Med 136:493–499. https://doi.org/ 10.1007/s00414-021-02748-z 11. Bowman Z, Daniel R, Gerostamoulos D et al (2022) Rapid DNA from a disaster victim identification perspective: is it a game changer? Forensic Sci Int Genet 58:102684. https:// doi.org/10.1016/j.fsigen.2022.102684 12. Watherston J, McNevin D, Gahan ME et al (2018) Current and emerging tools for the recovery of genetic information from post mortem samples: new directions for disaster victim identification. Forensic Sci Int Genet 37:270– 282. https://doi.org/10.1016/j.fsigen.2018. 08.016 13. Holland M, Wendt F (2015) Evaluation of the RapidHIT™ 200, an automated human identification system for STR analysis of single source samples. Forensic Sci Int Genet 14:76– 85. https://doi.org/10.1016/j.fsigen.2014. 08.010

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14. Date-Chong M, Hudlow WR, Buoncristiani MR (2016) Evaluation of the RapidHIT™ 200 and RapidHIT GlobalFiler® Express kit for fully automated STR genotyping. Forensic Sci Int Genet 23:1–8. https://doi.org/10. 1016/j.fsigen.2016.03.001 15. Thermo Fisher Scientific (2018) RapidLINK™ Software v1.0 user guide. Available via Thermo Fisher Scientific. https://assets.thermofisher. com/TFS-Assets/LSG/manuals/MAN001 8038_RapidLinkSW1_UG.pdf. Accessed 21 Apr 2022 16. Applied Biosystems (2021) RapidHIT™ ID System v1.3.1 user guide. Available via Thermo Fisher Scientific. https://assets.thermofisher. com/TFS-Assets/LSG/manuals/MAN001 8938_RapidHIT_ID_System_v1_3_1_UG. pdf. Accessed 21 Apr 2022 17. Gill P, Fereday L, Morling N et al (2006) The evolution of DNA databases—recommendations for new European STR loci. Forensic Sci Int 156:242–244. https://doi.org/10.1016/ j.forsciint.2005.05.036 18. Shackleton D, Gray N, Ives L et al (2019) Development of RapidHIT® ID using NGMSElect™ Express chemistry for the processing of reference samples within the UK criminal justice system. Forensic Sci Int 295: 179–188. https://doi.org/10.1016/j. forsciint.2018.12.015 19. Applied Biosystems (2019) RapidINTEL™ sample cartridge for blood and saliva samples validation user bulletin, revision A. Available via Thermo Fisher Scientific. https://assets. t h e r m o fi s h e r. c o m / T F S - A s s e t s / L S G / manuals/MAN0018979_RapidINTEL_ RHIT_v1_1_3_Validation_UB.pdf. Accessed 21 Apr 2022 20. Gomes C, Martı´nez-Go´mez J, Dı´ez-Jua´rez L et al (2017) Prep-n-Go™: a new and fast extraction method for forensic blood samples. Forensic Sci Int 6:e265–e266. https://doi. org/10.1016/j.fsigss.2017.09.089 21. Applied Biosystems (2014) GlobalFiler™ Express PCR Amplification Kit user guide, revision G. Available via Thermo Fisher Scientific. https://assets.thermofisher.com/TFSAssets/LSG/manuals/4477672_ GlobalFilerExpress_UG.pdf. Accessed 21 Apr 2022 22. Applied Biosystems (2018) AmpFlSTR™ Identifiler™ Direct PCR Amplification Kit user guide, revision K. Available via Thermo Fisher Scientific. https://assets.thermofisher. com/TFS-Assets/LSG/manuals/cms_0 65522.pdf. Accessed 21 Apr 2022 23. Romsos E, Vallone P (2015) Rapid PCR of STR markers: applications to human

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identification. Forensic Sci Int Genet 18:90– 99. https://doi.org/10.1016/j.fsigen.2015. 04.008 24. Kermekchiev M, Kirilova L, Vail E et al (2009) Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Res 37(5):e40. https://doi.org/10. 1093/nar/gkn1055 25. Spitaleri S, Piscitello D, di Martino D et al (2004) Experimental procedures comparing the activity of different Taq polymerases. Forensic Sci Int 146S:S167–S169. https:// doi.org/10.1016/j.forsciint.2004.09.051 26. Butler J (2012) Advanced topics in forensic DNA typing: methodology. Academic Press Elsevier, Waltham, MA 27. Verheij S, Harteveld J, Sijen T (2012) A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples.

Forensic Sci Int Genet 6:167–175. https:// doi.org/10.1016/j.fsigen.2011.03.014 28. Yang Y, Kim J, Song Y et al (2007) A novel buffer system, AnyDirect, can improve polymerase chain reaction from whole blood without DNA isolation. Clin Chim Acta 380:112– 117. https://doi.org/10.1016/j.cca.2007. 01.019 29. Bu Y, Huang H, Zhou G (2008) Direct polymerase chain reaction (PCR) from human whole blood and filter-paper-dried blood by using a PCR buffer with a higher pH. Anal Biochem 375:370–372. https://doi.org/10. 1016/j.ab.2008.01.010 30. Applied Biosystems (2015) AmpFlSTR® NGM SElect™ PCR Amplification Kit, revision F. Available via Thermo Fisher Scientific. https://tools.thermofisher.com/content/sfs/ manuals/cms_089008.pdf. Accessed 21 Apr 2022

Chapter 24 Next-Generation Sequencing: ForenSeq™ DNA Signature Prep Kit with the Illumina MiSeq FGx Megan M. Foley Abstract Sequencing forensic DNA samples that are amplified and prepared with the ForenSeq™ DNA Signature Prep Kit allows for the simultaneous targeting of forensically relevant STR and SNP markers. The MiSeq™ FGx system allows massively parallel sequencing of these markers in a single analysis. The library preparation targets autosomal, Y-, and X-STRs, as well as identity SNPs. The kit can also be used to generate investigative information regarding the DNA contributor by analyzing phenotypic SNPs to predict hair color, eye color, and ancestry SNPs. Through two rounds of amplification, all loci are amplified and tagged with individualizing barcodes for sequencing capture and identification. Using bead-based technology, the libraries are purified by the removal of left-over amplification reagents and then normalized to ensure equal representation of all samples during sequencing. The individual libraries are then pooled for insertion into the MiSeq FGx. The pooled libraries are then added to a pre-packaged cartridge that contains all reagents necessary for optimal sequencing. Libraries are captured on a flow cell and undergo bridge amplification for the generation of individual clusters. Sequencing of each cluster is performed using a Sequence-By-Synthesis technology. The following chapter describes the methodology and process of library preparation of samples using the ForenSeq™ DNA Signature Prep Kit Primer Set A and B. Once completed, the chapter then focuses on the setup of a sequencing run on the MiSeq FGx and the sequencing methodology employed by the instrument. Key words ForenSeq™ DNA Signature Prep Kit, MiSeq FGx, Forensic DNA Sequencing, Next Generation Sequencing, Massively Parallel Sequencing, STR Sequencing, SNP Analysis, Phenotypic SNPs, Ancestry SNPs

1 1.1

Introduction Background

Verogen’s ForenSeq™ DNA Signature Prep Kit is one of the first commercial sequencing assays manufactured for forensic purposes [1, 2]. In tandem with the Illumina MiSeq™ FGx instrument, this system allows for enhanced multiplexing capabilities by utilizing massively parallel sequencing (MPS) for common forensic short tandem repeats (STRs) and a variety of single nucleotide polymorphisms (SNPs) for analysis of crime scene and reference

Catherine Cupples Connon (ed.), Forensic DNA Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2685, https://doi.org/10.1007/978-1-0716-3295-6_24, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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Table 1 Primer Set A and Primer Set B locus specifications STRs

SNPs

Genetic marker type

Autosomal

Y

X

Identity

Ancestral

Phenotypic

Primer Set A

27

24

7

94

–

–

Primer Set B

27

24

7

94

56

22

Displayed is a breakdown of the number of genetic loci per target type in the Primer Set A and Primer Set B options. Two SNPs are common between the ancestral and phenotypic groupings

samples. Two separate data sets can be generated based on the sample type or a laboratory’s preference. Primer Set A can be used for identity or kinship purposes. A total of 58 STRs are targeted, including autosomal (including Amelogenin), Y-, and X-STRs; and a total of 94 identity SNPs. Primer Set B is an alternative to set A that can be used for identity purposes—using the same 152 markers as Set A—and to generate possible investigative leads based on predicted phenotypic features, including hair and eye color, as well as predicted ancestral features, using an additional 78 SNPs (see Tables 1 and 2) [1–4]. The Verogen system allows for a large increase in the amount of genetic information that can be gathered from a forensic or reference sample compared to the typical genetic profile generated from a megaplex STR kit designed for capillary electrophoresis (CE) detection. Through current CE procedures, the fragment size of the STR repeat is reported and utilized for analysis. Sequencing allows forensic analysts to observe and utilize the entire sequence of the STR repeat, which allows for the identification of isoalleles. Isoalleles can be observed when two fragments of DNA have the same length but are different in sequence. Current CE methods are unable to detect this sequence variation, making isoalleles indistinguishable from one another on that detection platform. Additionally, through the identification of isoalleles, it is possible to separate out stutter fragments that are the same length as minor contributors with differing sequences. Following current analysis and interpretation methods, stutter filter percentages and peak height ratios only indicate the possible stacking of stutter peaks with low level contributors. Sequencing can allow for further verification of this occurrence in a profile, which allows for more accurate DNA interpretation when deducing mixtures or comparing a reference to a mixture profile [2]. Although not currently used for forensic casework purposes, the ForenSeq™ kit also has the ability to sequence flanking regions for identity or comparison purposes in the future, which also can add additional variability to a profile [5]. Additional benefits of the kit include the ability to sequence the above-

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Table 2 ForenSeq™ Signature Prep Kit targeted loci DNA target

List of loci

Autosomal STR D1S1656, TPOX, D2S441, D2S1338, D3S1358, D4S2408, FGA, D5S818, loci CSF1PO, D6S1043, D7S8201, D8S1179, D9S1122D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D17S1301, D18S51, D19S433, D20S482, D21S11, Penta D, D22S10452 Y-STR loci

DYF387S1, DYS19, DYS385a-b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS437, DYS438, DYS439, DYS448, DYS460, DYS481, DYS505, DYS522, DYS533, DYS549, DYS570, DYS576, DYS612, DYS635, DYS643, Y-GATA-H4

X-STR loci

DXS10074, DXS10103, DXS10135, DXS7132, DXS7423, DXS8378, HPRTB

Identity SNPs

rs10495407, rs1294331, rs1413212, rs1490413, rs560681, rs891700, rs1109037, rs12997453, rs876724, rs907100, rs993934, rs1355366, rs1357617, rs2399332, rs4364205, rs6444724, rs1979255, rs2046361, rs279844, rs6811238, rs13182883, rs159606, rs251934, rs338882, rs717302, rs13218440, rs1336071, rs214955, rs727811, rs321198, rs6955448, rs737681, rs917118, rs10092491, rs2056277, rs4606077, rs763869, rs1015250, rs10776839, rs1360288, rs1463729, rs7041158, rs3780962, rs735155, rs740598, rs826472, rs964681, rs10488710, rs1498553, rs2076848, rs901398, rs10773760, rs2107612, rs2111980, rs2269355, rs2920816, rs1058083, rs1335873, rs1886510, rs354439, rs1454361, rs4530059, rs722290, rs873196, rs1528460, rs1821380, rs8037429, rs1382387, rs2342747, rs430046, rs729172, rs740910, rs8078417, rs938283, rs9905977, rs1024116, rs1493232, rs1736442, rs9951171, rs576261, rs719366, rs1005533, rs1031825, rs1523537, rs445251, rs221956, rs2830795, rs2831700, rs722098, rs914165, rs1028528, rs2040411, rs733164, rs987640

Ancestral SNPs

rs2814778, rs3737576, rs7554936, rs10497191, rs1834619, rs1876482, rs260690, rs3827760, rs6754311, rs798443, rs12498138, rs1919550, rs1229984, rs3811801, rs4833103, rs7657799, rs7722456, rs870347, rs16891982, rs192655, rs3823159, rs917115, rs1462906, rs1871534, rs2196051, rs6990312, rs3814134, rs4918664, rs1079597, rs174570, rs2238151, rs671, rs1572018, rs2166624, rs7326934, rs7997709, rs9522149, rs200354, rs12439433, rs1426654, rs1800414, rs735480, rs12913832, rs459920, rs11652805, rs17642714, rs2593595, rs4411548, rs4471745, rs2042762, rs3916235, rs4891825, rs7226659, rs7251928, rs310644, rs2024566

Phenotypic SNPs

rs28777, rs12203592, rs4959270, rs683, rs1042602, rs1393350, rs12821256, rs12896399, rs2402130, rs1800407, N29insA, rs1110400, rs11547464, rs1805005, rs1805006, rs1805007, rs1805008, rs1805009, rs201326893_Y152OCH, rs2228479, rs885479, rs2378249

All loci targeted by either Primer Set A and/or B are broken down by DNA target type [1]

mentioned markers in one analysis, which would require more time, money, analyses, and sample volume to perform on the CE [6]. Also, because of the decreased size of the STR fragments, and especially because of the minimal sizes of the identity SNP markers, more information can be gathered from a degraded sample [2, 7– 10].

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1.2 Library Preparation Using the ForenSeq™ DNA Signature Prep Kit

Sample types that can be processed using the ForenSeq™ kit include purified extracts that have been previously extracted and quantified, crude lysates that have undergone a direct quantification process, and FTA® Card punches. The procedure includes two amplification steps, a bead-based purification step, a bead-based normalization step, and pooled library preparation for sequencing. Altogether, it takes around 9 h total of processing and hands on time. The first round of amplification is similar to CE-based procedures and targets the STRs and SNPs to be sequenced utilizing oligonucleotide primers that surround the targeted DNA sequence. The primers contain forward and reverse tags to identify the amplified strands in later processes. The second round of amplification additionally enhances these targets and adds indexed adapters to each sequence that are complementary to the tag sequence added to the DNA fragments during Amplification 1 [1]. Later in the process, the samples and controls are combined into one tube as a pooled library. In order to separate out the sequenced fragments, each sample needs to be uniquely labeled. The reaction for Amplification 2 includes the addition of two indices, Index 1 (i7) and Index 2 (i5). Each index fragment contains two parts: a unique index sequence and a common adapter sequence. The indices are utilized to provide a unique combination of various i7 and i5 index pairings for each sample and allow for the sample multiplexing capacity that sequencing allows. Each index is made up of a unique combination of eight base pairs. The specific indices link with the sample name during run setup and act as a unique barcode, which allows the instrument to identify which sequences belong to each sample and separate data that belongs to that specific sample. The adapter sequences (120 bp) are identical on each fragment, regardless of the index portion of the tag, and are utilized for capture purposes for sequencing on the flow cell. The unique combination and the sample name are imported into the sequencing instrument for bioinformatics purposes (see Fig. 1) [1, 11]. After the second amplification step, samples need to be purified in order to remove any leftover amplification reagents that interfere with sequencing (e.g., leftover nucleotides, primers, etc.). The purification step utilizes magnetic beads that attract and bind to the DNA. The beads are removed from the solution through the use of a magnetic stand, leaving the DNA-free supernatant and any leftover reagents, which are subsequently removed and discarded. The beads are then washed twice utilizing an ethanol-based wash procedure in order to ensure a pure sample. After the wash steps have been performed and all residual ethanol is removed, a resuspension buffer is added to the samples that release the DNA from the magnetic beads, releasing it back into the solution. The magnetic stand can be used once again to draw the beads to the side, but this time the supernatant containing the DNA can be removed and further processed [1].

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Fig. 1 A representation of products from Amplification 1 and 2. The amplicon from Amplification 1 contains the amplified STR sequence (dark blue), flanking regions (green), forward and reverse primers (yellow), and the forward and reverse primer tags (orange for i5 and purple for i7). Amplification 2 includes the addition of i5 and i7 Index/Adapter strands. The i5 indices start with a region complementary to the forward primer tag and the i7 indices start with a region complementary to the reverse primer tag and binds (orange for i5 and purple for i7). The next section will contain the unique sequence specific to the index added to the sample (blue). Lastly, the indices have an i5 and i7 adapter sequence that is complementary to a stationary oligonucleotide on the flow cell and binds during sequencing

This library preparation procedure does not require the quantification of individual libraries but instead utilizes a DNA concentration normalization step with beads. Similar to the purification step, magnetic beads are added at equal concentrations, initially attracting and binding DNA. The beads for this step have a binding capacity, which limits the amount of sample added to the flow cell for sequencing. This is to create a pool of equally represented samples by limiting the amount of DNA library for larger quantity samples so that they do not overshadow any lower-level samples. The washing procedure is performed twice and utilizes a mixture that includes formamide and 2-mercaptoethanol (aka β-mercaptoethanol) [12]. A resuspension reagent is added to remove the DNA from the beads and the DNA is released back into solution. The liquid containing the DNA is removed for the next step [1]. The last step before preparing the sample for processing is the pooling of the libraries for sequencing. The number of samples that can be processed on one flow cell depends on the primer set used and the size of the flow cell (see Table 8) [1, 13]. Verogen manufactures two flow cell types that can be utilized in conjunction with the ForenSeq™ kit: the Standard Flow Cell and the Micro Flow Cell. All reagents are identical between the two flow cell kits and both perform a total of 600 sequencing cycles. The standard flow cell allows for more samples to be processed at once (up to 96 samples from Primer Set A or 32 samples from Primer Set B) and takes around 30 h because of the increase in fluorescence imaging time. The micro flow cell is best for laboratories that do not intend to have a large amount of samples processed using sequencing (up to

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36 samples from Primer Set A or 12 samples from Primer Set B), which allows for decreased processing time (reduced by ~6 h) and a decrease in cost per sample [13]. To prepare the pooled libraries for sequencing, it must first undergo a denaturation process similar to CE-based methods. The pooled libraries are first diluted out in a hybridization buffer. At this stage, a sequencing control is added that is used by the instrument’s software for quality checks and can be used to determine if the run was successful or may contain errors. Denaturation of DNA occurs through a heating process and a snap-cool procedure. Once this process is complete, the sample can be loaded into a sequencing reagent cartridge and loaded into the instrument. Each cartridge is single use and contains all necessary reagents required for cluster generation [1, 11]. 1.3 Sequencing on the MiSeq FGx™

Sequencing on the Illumina MiSeq FGx™ occurs on a glass flow cell and can be broken down into multiple stages. The first is bridge-amplification to form amplified clusters. The flow cell contains oligonucleotides bound to the bottom. These oligonucleotides are complementary to the adapters that are attached to each DNA fragment during the index/adapter addition in Amplification 2. Each fragment binds to the flow cell through these adapters. The adapter on the reverse side of the fragment bends over and binds to an additional oligonucleotide. All DNA fragments are now bound at both ends and forms an upside-down U shape or a “bridge.” Polymerase enzymes and nucleotides are flushed through the flow cell. Through the addition of a primer, each strand is replicated. One adapter on each fragment (the template and the new amplified strand) will be released and two identical strands are present. During the next cycle, both strands bend over to form a new bridge and the amplification repeats. This occurs over and over again until individual clusters of each amplified product are formed. Each cluster contains 1000+ of copies of one DNA target for one unique amplicon. Since we have undergone multiple rounds of amplification, we expect each unique DNA target to be present in multiple clusters, which allows for optimal sequencing reads and results [11]. Next, the amplified clusters are sequenced through a process called “Sequencing-By-Synthesis” or SBS. A new primer attaches to each fragment that is once again complementary to the adapter. The instrument floods the flow cell with a mixture of all four dideoxynucleotides that are fluorescently labeled with different molecules based on nucleotide. A blocking agent present will restrict the addition of another nucleotide to the growing strand, which allows detection of stretches of the same nucleotide. The instrument then uses an imaging system to capture the fluorescence of the dye. The wavelength that is captured by the system indicates which nucleotide has been added. The imaging process

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uses various combinations of LED and filters and occurs multiple times during each cycle. A flow cell is broken up into different imaging sections, or tiles, and each tile is imaged separately. The standard flow cell is made up of 19 tiles, while the micro flow cell has eight, leading to a decrease in processing time by ~6 h [11, 13]. A deblocking agent is then flooded over the flow cell to expose the previously attached nucleotide for the next base pair addition. This occurs over and over until both indices, the flanking region, the forward and reverse tags, and target regions have been sequenced. After image analysis, the software performs a series of bioinformatic analyses that includes base calling, filtering, and calculating a quality score for each strand. Further analysis on the genotypes is performed in the ForenSeq™ Universal Analysis Software for forensic analysis [11, 14].

2

Materials For pipetting reagents and samples, utilize aerosol-barrier pipette tips. Any plastics utilized should be RNase/DNase free (microcentrifuge tubes, conical tubes, reagent reservoirs, etc.). The temperature ranges for storage indicated for each reagent include: refrigerator (2 to 8 °C), freezer (-25 to -15 °C), and room temperature (15 to 30 °C).

2.1 Library Preparation

1. Sample sources: acceptable types of samples for this assay include purified DNA, crude lysate, or blood/saliva on FTA® Cards (see Note 1). 2. Pipettes: multi-channel [8], single channel, and repeater pipettes, plus corresponding tips (see Note 2). 3. PCR tubes: 8-tube strips and caps. 4. 96-well 0.3 mL skirted or semi-skirted PCR plates. 5. 96-well storage plates: round well, 0.8 mL; also referred to as a “midi plate.” 6. Disposable reagent reservoirs for multi-channel pipettes. 7. Microseal “A” film (see Note 3). 8. Microseal “B” adhesive seals (see Notes 3). 9. Index Adapter Replacement Caps. 10. 200 proof (absolute) ethanol: molecular-biology grade. 11. Water: nuclease-free, molecular-biology grade. 12. FTA® Card extraction buffer (see Notes 4 and 5). 13. 1X Tris-Borate-EDTA (TBE) buffer (see Note 4). 14. ForenSeq™ DNA Signature Prep Kit: available as a 96 or 384 reaction kit.

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15. ForenSeq™ DNA Signature Prep Kit—Pre-Amplification 1 Reagents: contains 2800M Control DNA, DNA Primer Mix A (DPMA), DNA Primer Mix B (DPMB), Enzyme Mix (FEM), and PCR1 Reaction Mix (PCR1). Store all components in a freezer upon receipt. 2800M Control DNA can be stored in a refrigerator after the first use. 16. ForenSeq™ DNA Signature Prep Kit—Pre-Amplification 2 Reagents: contains PCR2 Reaction Mix, i7 index orange capped tubes (12 total), i5 index white capped tubes (8 total), and additional i7/i5 index tube caps. Store all components in a freezer upon receipt, except the additional tube caps. 17. ForenSeq™ DNA Signature Prep Kit—Purification Reagents: contains Resuspension Buffer (RSB) and Sample Purification Beads (SPB). Store all components in a refrigerator upon receipt. 18. ForenSeq™ DNA Signature Prep Kit—Normalization Reagents: contains HP3 (2 N NaOH), Library Normalization Additives 1 (LNA1), Library Normalization Beads 1 (LNB1), Library Normalization Storage Buffer 2 (LNS2), and Library Normalization Wash 1 (LNW1) (see Note 6). Store all components in a freezer upon receipt. LNB1, LNS2, and LNW1 can be stored in a refrigerator after the first use. 19. ForenSeq™ DNA Signature Prep Kit—Denaturation/Dilute Reagents: contains Human Sequencing Control (HSC) and MiSeq FGx™ Reagent Kit (Hybridization Buffer (HT1) and Reagent Cartridge). Store all components in a freezer upon receipt. 20. Plate seal applicator. 21. 1.2 mm FTA® Card punching tool (see Note 4). 22. 96-well plate base (see Note 7). 23. ForenSeq™ Index Plate Fixture. 24. Magnetic stand (see Note 8). 25. 1.5 mL tube benchtop cooler. 26. 1.5 mL 96-well micro-heating system/heat block. 27. High-speed thermal mixers (see Note 9). 28. 96-well thermal cycler: must be approved by Verogen (see Table 4). 29. 96-well plate shaker (see Note 9). 2.2 Sequencing Specific Materials

1. MiSeq disposable wash tubes. 2. Water: nuclease-free, molecular-biology grade. 3. 6% Sodium Hypochlorite.

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4. MiSeq FGx Reagent Kit: This 380-cycle kit comes in two sizes—standard and micro. The standard kit can process 96 samples with Primer Set A and 32 samples with Primer Set B; the micro kit can process 36 samples with Primer Set A and 12 samples with Primer Set B. Each kit contains Hybridization Buffer (HT1), Reagent Cartridges, Flow Cell Containers, and SBS Solution (PR2); the first two components are stored in the freezer, while the latter two are stored refrigerated. 5. 1.5 mL tube benchtop cooler. 6. Large volume repeat pipettor (>5 mL) and appropriate tips.

3

Methods The complete NGS process is a very lengthy procedure; be sure to allot ample time for each portion of the assay (see Table 3). Before beginning each section of the method, ensure that all reagents are within expiration and that all necessary equipment and/or consumables are stocked. Prepare all worksheets with appropriate information (e.g., sample names, assigned index adapters, extract volumes needed, master mix calculations, etc.). Any extraction

Table 3 Time and storage conditions following each step of the NGS process

Process

Total time to Handscomplete on time

Okay to pause after completion?

Amplification 1: amplification and tagging of loci targets

3 h 35 min

15 min

Yes, amplification product can be stored at 2–8 °C for up to 2 days.

Amplification 2: enrichment of targets

1 h 30 min

10 min

Yes, amplification product can be stored at 2–8 °C for up to 7 days.

Sample purification

30 min

15 min

Yes, purified samples can be stored at -25 to -15 °C for up to 1 year.

Sample normalization

1 h 20 min

30 min

Yes, normalized samples can be stored at -25 to -15 °C for up to 30 days.

Pooling of sample libraries

10 min

10 min

Yes, pooled samples can be stored at -25 to -15 °C for up to 30 days.

Denaturation and dilution of pooled libraries

10 min

10 min

No, immediately proceed to instrument setup and performing a run.

Instrument setup and performing 10 min a run

10 min

N/A

The overall process of next-generation sequencing is lengthy. The entire process can be completed in one day or it can be broken up into smaller sections. This table outlines where natural breaks occur, whether the process can be paused after these individual steps, and if so, how to store the samples before proceeding. If pausing after a step that leaves the samples in a 96-well format, ensure that the plate is securely sealed with a Microseal “B” or cap strips

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controls should be processed alongside samples. A sequencing amplification positive and negative control should be prepared. Additionally, a sequencing control should be added when sequencing the samples. Batch samples with similar primer targets and sample type (i.e., reference samples, high quality reference-like samples, and low quantity samples). While performing each section, it is crucial to avoid chances of contamination and decrease pipetting variability between samples. Pipette tips should be aerosol-resistant and should be changed between reagents, samples, and rows/columns if using a multichannel pipette. With each aspiration using a multi-channel pipette, ensure that equal volumes of liquid are present in each tip, since some variability can exist between the channels. Additionally, reagents and consumables for Amplification 1 should be stored in pre-amplification (pre-amp) work areas. Preparation for Amplification 1 should also be performed in pre-amp; all other subsequent steps should be performed in post-amplification (post-amp) work areas to prevent contamination of pre-amp areas. 3.1 Library Preparation—Amplification 1: Amplification and Tagging of Loci Targets

1. Thaw all necessary Amplification 1 reagents to room temperature (~30 min for the 2800M standard) (see Note 10). 2. Label a 96-well plate “FSP” for ForenSeq™ Sample Plate (see Notes 11 and 12). 3. Create a thermal cycler program for Amplification 1 (see Tables 4 and 5). The total amplification time is ~3.5 h.

Table 4 Verogen recommended thermal cyclers and amplification settings Ramp mode for amp 1 and 2

Lid temperature settings

Temperature mode settings

Veriti™ 96-well Thermal Cycler

4%

Heated at a constant temp of 105 °C

Standard

ProFlex™ 96-well PCR System

0.2 °C per second Heated at a constant temp of 105 °C

None Provided

GeneAmp PCR System 9700a

8%

Heated

9600 emulation

Bio-Rad

4%

Heated at a constant temp of 100 °C

Calculated

Eppendorf® Mastercycler® Pro S

2%

Heated

Gradient S, Simulated tube

Thermal cycler

Other notes

Only supports gold heat block

Verify that ramp mode and temperature settings match the listed thermal cycler type. All thermal cyclers allow for plates and tubes made of polypropylene, except the Eppendorf® Mastercycler® Pro S, which only allows for plates a Also referred to as the ABI LTI Thermal Cycler 9700

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Table 5 Amplification 1 and 2 thermal cycler parameters Step

Amplification 1

Amplification 2

Preheat lid option

100 °C

100 °C

Initial heat

98 °C for 3 min

98 °C for 30 s

PCR cycling

8 cycles of: 96 °C for 45 s 80 °C for 30 s 54 °C for 2 min* 68 °C for 2 min*

15 cycles of: 98 °C for 20 s 66 °C for 30 s 68 °C for 90 s*

PCR cycling

10 cycles of: 96 °C for 30 s 68 °C for 3 min*

Final extension

68 °C for 10 min

68 °C for 10 min

Indefinite hold

10 °C

10 °C

Each step is broken down into temperature, duration, and cycle numbers. For steps marked with an “*”, ensure the ramping mode chosen is the appropriate mode for the thermal cycler based on Table 4

Amplification 1 can be run during the day or overnight depending on the workflow of the laboratory. 4. Prepare the samples for purified DNA extracts (see Subheading 3.1, step 5) or FTA® Cards (see Subheading 3.1, steps 6–8). Crude lysate samples do not require additional preparation. 5. For purified DNA, the manufacturer recommends a total of 1 ng in 5 μL total human DNA for optimal sequencing results (see Note 1). Using the quantitation values in ng/μL, calculate the appropriate volume of extract and nuclease-free water required to create a dilution with a final concentration of 0.2 ng/μL (see Note 13). These prepared samples will be added to the amplification plate after the master mix has been dispensed (see Subheading 3.1, step 15). Proceed to prepare the positive control (see Subheading 3.1, step 9). 6. For FTA® Card samples, use a single 1.2 mm punch of the dried stain from the card, ensuring to follow laboratories decontamination procedures after each punch is taken. 7. Aliquot 100 μL 1X TBE buffer to each well. Seal the plate with Microseal “B” or cap strips. Place the 96-well plate on an appropriately sized rack and shake for 2 min at 1800 rpm. 8. Centrifuge the plate using a plate spinner for 30 s at 1000 × g. Carefully remove the seal or caps. Remove and discard all supernatant using a 100 μL multi-channel pipette.

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Table 6 Composition of Amplification 1 reaction for various sample types for library preparation FTA® Cards Component

Purified DNA

Crude lysate

Samples ®

Controls

DNA input

5.0 μL (1 ng)

2.0 μL

FTA punch

5.0 μL (1 ng)

Master mix total

10.0 μL

13.0 μL

15.0 μL

10.0 μL

PCR1

4.7 μL

4.7 μL

4.7 μL

4.7 μL

FEM

0.3 μL

0.3 μL

0.3 μL

0.3 μL

DPMA or DPMB

5.0 μL

5.0 μL

5.0 μL

5.0 μL

Nuclease-free water

–

3.0 μL

5.0 μL

–

Total reaction volume

15.0 μL

15.0 μL

15.0 μL

15.0 μL

The composition of the Amplification 1 PCR reaction varies slightly based upon the sample type being processed, but the overall reaction volume for all is 15 μL, with a target of 1 ng of DNA for purified DNA and the positive amplification control

9. Similar to purified DNA samples, 1 ng of DNA is targeted for the amplification of the positive control, but the volume allotted is dependent on the type of sample being processed (see Table 6). To prepare the positive control, begin by vortexing and pulse spinning the 2800M Control DNA. If processing purified DNA and/or FTA® Card samples, dilute 2800M with nuclease-free water to a final concentration of 0.2 ng/μL. If processing crude lysate, dilute 2800M to 0.5 ng/μL. The diluted 2800M positive control will be added to the amplification plate after the master mix has been dispensed (see Subheading 3.1, step 15). 10. The composition of the amplification master mix depends on the type of sample being processed (see Table 6). The volume needed for each component should be multiplied by the total number of samples and controls (including extraction reagent blanks, as well as a positive and negative amplification control), plus an additional 10% for pipetting error (see Notes 14–16). 11. Label an appropriately sized microcentrifuge tube (e.g., 1.5 mL or 2.0 mL) as “Master Mix.” 12. Before pipetting PCR1 or DPMA/B, be sure to vortex these tubes and pulse spin them to remove any liquid from the cap. Do not vortex FEM, which is unstable. Instead, using a 100 μL pipette, pipette the liquid up and down gently to mix before adding the appropriate amount into the master mix. 13. Once all components have been added, pipette the master mix to mix and pulse spin to remove any liquid from the cap.

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14. Add the appropriate master mix volume per well based on the sample type (see Table 6). Depending on the capabilities of the lab, the master mix can be distributed into each well multiple ways (see Note 17). 15. Next, add prepared purified DNA samples (see Subheading 3.1, step 5) and/or crude lysate samples (if processing), as well as the amplifications controls, to the corresponding wells of the 96-well plate already containing master mix; if processing FTA® punches, these should already have been added (see Subheading 3.1, steps 6–8). Use nuclease-free water for the amplification negative control. Vortex and pulse spin all samples/controls before aliquoting. Specific volumes to be added for each are dependent on the sample type being processed (see Table 6). Once added, flush the tip to mix (see Note 18). 16. Seal the plate with a Microseal “A” using a plate seal applicator or cap strips for amplification (see Note 19). For fewer samples, cap strips can be utilized or Microseal “A” strips can be cut to fit the plate. Microseal “B” seals should not be used during thermal cycling. 17. Using a plate centrifuge, spin the plate for 30 s at around 1000 × g. 18. Transfer the plate to the post-amplification area and run the pre-programmed amplification as defined for Amplification 1 (see Table 5). 19. The plate can be left on the thermal cycler overnight. The expiration of the plate is 2 days. It can be processed immediately for the second amplification (see Subheading 3.2, step 1) or stored in a refrigerator (2–8 °C) until ready to proceed. If storing, remove Microseal “A” and replace with a Microseal “B” or strip caps. Microseal “B” or strips can be cut to fit the plate. 3.2 Library Preparation—Amplification 2: Enrichment of Targets— Amplification and Attachment of Indices

1. Thaw all necessary Amplification 2 reagents to room temperature (~20 min for the adapters) (see Note 10). 2. Remove the FSP plate from the thermal cycler or refrigerator (if stored) (see Subheading 3.1, step 19) and allow to come to room temperature. Cross out any labeling on the plate to ensure no confusion and relabel the plate as “FSP2” for ForenSeq™ Sample Plate Amplification 2 (see Notes 11 and 12). 3. Create a thermal cycler program for Amplification 2 (see Table 4 and Table 5). The total amplification time is ~1 h. Amplification 2 can be run during the day or overnight depending on the workflow of the laboratory. 4. Centrifuge the FSP2 plate for 30 s at 1000 × g to remove any liquid that has gathered on the seal or caps.

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5. To set up the ForenSeq™ Index Plate Fixture, begin by vortexing and pulse spinning the Index 1 (i7) and Index 2 (i5) tubes (see Note 20). 6. Place the i7 tubes (orange caps) within the appropriate column holders (1–12 of a 96-well plate) of the plate fixture and the i5 (white caps) within the appropriate column holders (A–H of a 96-well plate) of the plate fixture (see Notes 21 and 22). 7. Gently unscrew each of the i7 caps to the point where it can just be lifted off but do not remove. 8. Place the FSP2 plate in the center of the plate fixture (see Note 23). Carefully remove and discard the seal or cap strips. 9. Once all index tube caps are unscrewed, remove and discard the i7 caps (orange). To decrease the chance of contamination, start on one side of the plate to remove the caps and work across the row without reaching over an open tube or plate. 10. Aspirate 4 μL Index 1 (orange i7) into each row using a multichannel pipette. Check all tips after drawing up the liquid to ensure that equal volumes are present before adding to the wells. Pipette into the bottom of the well. Dispose of the used tips with every row. Cap each i7 tube with a new orange cap (see Note 24). Check that each well has a yellow tint (due to the i7 index reagent) and appears to contain the same volume, which can be viewed through the bottom of the plate. 11. Gently unscrew, remove, and discard the i5 caps (white). To decrease the chance of contamination, start on one side of the plate to remove the caps and work across the row without reaching over an open tube or plate. 12. Aliquot 4 μL Index 2 (white i5) into each column using a multi-channel pipette. Check all tips after drawing up the liquid to ensure that equal volumes are present before adding to the wells. Pipette into the bottom of the wells. Dispose of the used tips with every column. Cap each i5 tube with a new white cap (see Note 24). Looking at the underside/bottom of the plate, check that each well appears to contain the same volume. 13. Vortex and pulse spin PCR2. Aliquot 27 μL into each well using a separate pipette tip for each sample (see Note 25). 14. Seal the plate with a Microseal “A” using a plate seal applicator or cap strip for amplification (see Note 19). 15. Using a plate centrifuge, spin the plate for 30 s at around 1000 × g. 16. Amplify the plate using the pre-programmed amplification as defined for Amplification 2 (see Table 5).

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17. The plate can be left on the thermal cycler overnight. The expiration of the plate is 7 days. It can be processed immediately to purify the samples (see Subheading 3.3, step 1) or stored in a refrigerator (2–8 °C) until ready to proceed. If storing, remove Microseal “A” and replace with a Microseal “B” or cap strips. 3.3 Library Preparation—Sample Purification—Removal of Left-over Amplification Reagents

1. Thaw all necessary Purification Reagents to room temperature (~30 min for both SPB and RSB). Make sure to allow sufficient time before beginning to allow all contents to thaw and resolubilize (see Note 10). 2. Remove the FSP2 plate from the thermal cycler or refrigerator (if stored) (see Subheading 3.2, step 17) and allow to come to room temperature. 3. Label a new midi plate “PBP” for Purification Bead Plate (see Note 12). 4. To prepare the Sample Purification Beads (SPB) and midi plate, begin by vortexing the beads thoroughly (see Note 26). When pipetting, aspirate and dispense gently to ensure the appropriate amount of beads are drawn up/dispensed (see Note 27). 5. Calculate the volume of SPB based on the number of samples/ controls being processed (see Table 7). Vortex frequently to ensure equal distribution of beads. Pipette 45 μL into each well of the PBP midi plate with a single channel or multi-channel. Ensure that the liquid is dispensed directly into the bottom of the well with no liquid on the side.

Table 7 Volume of SPB needed for sample purification # Samples/ Volume of SPB (μL) Comments controls 96

(# samples/controls × 50) + 200

Add the entire volume of SPB to a reagent reservoir and then use a multi-channel pipette to transfer to the wells of the PBP midi plate

It is essential that the correct volume of SPB is added to each sample during the sample purification process. As with many routine laboratory procedures, additional reagent should be included in the aliquot to account for pipetting error, and in this case, the mode of delivery to the samples (e.g., single-channel vs multi-channel pipette with reagent reservoir) also impacts how much overrage to include. This table serves as a guide for the amount of SPB overrage likely needed for this procedural step but should be adjusted as needed for individual practice

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6. Centrifuge the FSP2 plate for 30 s at 1000 × g to remove any condensation from the seal or caps. 7. Using a multi-channel pipette, transfer 45 μL of each sample/ control from the FSP2 plate to the corresponding wells of the PBP midi plate. Ensure that the liquid is dispensed directly into the bottom of the well with no liquid on the side (see Note 28). The FSP2 plate will not have enough volume for additional processing and can be discarded. 8. Seal the PBP midi plate with a Microseal “B” using a plate sealer and shake for 2 min at 1800 rpm. 9. Once shaking is complete, let the plate sit at room temperature for 5 min (no shaking). 10. Place the PBP midi plate on the magnetic stand. Carefully remove the seal. Let the plate sit for 2 min or until the liquid is clear and all beads have gathered toward the magnet. Beads gather in opposite directions every other row based on where the magnet is located (see Note 29). 11. Using a 100 μL multi-channel pipette, remove and discard any supernatant from each well (see Note 30). To avoid disrupting the beads, insert the pipette into the opposite side of the beads (plunger pressed down). Touch the tips to the opposite side at the top of the well and slowly move the tips downwards until it reaches the bottom. Once at the bottom, gently straighten the pipette tip. Lift up gently and slowly aspirate the liquid (see Note 31). Discard the liquid. 12. Prepare 440 μL fresh 80% ethanol (EtOH) per sample/control in a 15 mL or 50 mL conical tube (see Note 32). Vortex or invert to mix thoroughly and then pour the ethanol into a reagent reservoir. 13. While the PBP midi plate remains on the magnetic stand, perform an EtOH wash two times. Using a multi-channel pipette, add 200 μL 80% EtOH into each well. Let the plate incubate for 30 s on the magnetic stand. Remove and discard all supernatant with a multi-channel pipette; use clean tips for each column. Repeat for the second wash. 14. Seal the midi plate with a Microseal “B” and centrifuge for 30 s at 1000 × g. This should draw any leftover EtOH to the bottom of the wells. 15. Carefully remove the seal and place the PBP midi plate back on the magnetic stand. Let the plate sit until beads have once again accumulated toward the magnet. 16. Remove and discard any leftover liquid from each well using a 20 μL multi-channel pipette. Check that no liquid remains at the bottom of the plate (see Note 33). If liquid is still present, individual wells can be targeted using a single-channel pipette.

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17. Invert the Resuspension Buffer (RSB) conical tube a few times and remove the PBP midi plate from the magnetic stand. 18. If using a multi-channel pipette to dispense RSB to each sample, multiply the amount of samples/controls by 58 μL (includes extra for pipetting error). Pipette this amount of RSB into a reagent reservoir and then pipette 52.5 μL into each applicable well of the PBP midi plate. Otherwise, for a small number of samples/controls, use a single-channel pipette to add 52.5 μL to each applicable well. Check that each well is holding equal volumes after the addition. 19. Seal the plate with a Microseal “B” using the plate sealer and shake for 2 min at 1800 rpm. After 2 min has elapsed, check that all beads have been resuspended in each well. If not, the shake may be repeated, or individual wells can be mixed using a pipette. 20. Let sit for 2 min with no shaking. Place the PBP midi plate on the magnetic stand. Carefully remove the seal. Let sit for 2 min or until the liquid is clear and all beads have gathered towards the magnet. 21. Label a new 96-well plate “PLP” for Purified Library Plate (see Notes 11 and 12). Using a multi-channel pipette, remove 50 μL of each sample from the PBP midi plate and transfer to the corresponding well of the new PLP 96-well plate (see Note 34). 22. Seal the PLP plate with Microseal “B” or cap strips and centrifuge for 30 s at 1000 × g. 23. The expiration of the purification plate is 1 year. It can be processed immediately to normalize the samples (see Subheading 3.4, step 1) or stored in a freezer (-25 to -15 °C) until ready to proceed. 3.4 Library Preparation—Sample Normalization—To Create Equal Sample Representation During Sequencing

1. Thaw all necessary reagents to room temperature (LNB1 and LNW1 will take longer than the other reagents, at ~30 min total for both). Make sure to allow sufficient time before beginning (see Note 10). 2. Continue processing the room temperature PLP plate or if stored, remove from the freezer and allow to come to room temperature (see Subheading 3.3, step 23). 3. Label a new midi plate “NWP” for Normalized Working Plate (see Note 12). 4. To prepare the LNA1/LNB1 master mix, begin by vortexing the LNB1 beads thoroughly for at least 1 min, and invert at least 5 times every 15 s until beads are aspirated to the master mix tube (see Notes 26, 27, and 35).

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5. Calculate the volume needed of LNA1 and LNB1 for the master mix by multiplying the number of samples/controls by 46.8 μL and 8.5 μL, respectively (these volumes include extra for pipetting error). 6. Label an appropriately sized tube as “Master Mix” and add the calculated volumes of LNA1 and LNB1. When pipetting LNB1, aspirate and dispense gently to ensure the appropriate volume of beads is drawn up (see Note 27). 7. Vortex the master mix and invert to thoroughly mix the beads. Immediately pipette the contents into a reagent reservoir using a 1000 μL pipette (see Note 36). 8. Transfer 45 μL master mix into each well of the midi plate using a 100 μL or similar multi-channel pipette (see Notes 35). Ensure that the liquid is dispensed directly into the bottom of the well with no liquid on the side (see Note 37). To maintain homogeneity of the master mix in the reagent reservoir, gently pipette the remaining master mix up and down a few times in between the addition of master mix to each column of the midi plate. 9. Centrifuge the PLP plate for 30 s at 1000 × g to remove any condensation from the seal or caps. 10. Place the PLP plate on the magnetic stand. Carefully remove the seal or caps. Let sit for 2 min or until the liquid is clear and all beads have gathered towards the magnet (see Note 38). 11. Using a multi-channel pipette, transfer 20 μL of the samples from the PLP plate to the corresponding wells of the NWP midi plate. Ensure that the liquid is dispensed directly into the bottom of the well with no liquid on the side (see Note 37). Do not discard the PLP plate. There is enough volume for an additional normalization process. Re-seal with Microseal “B” or strips caps and place the PLP plate back into the freezer for storage. 12. Seal the NWP midi plate with a Microseal “B” using the plate sealer and shake for 30 min at 1800 rpm. 13. Preparation of 0.1 N HP3 and the NLP plate can be performed during the NWP incubation. 14. To prepare 0.1 N HP3 reagent, begin by calculating the volume needed of nuclease-free water and HP3 by multiplying the number of samples/controls by 33.3 μL and 1.8 μL, respectively (these volumes include extra for pipetting error). 15. Label an appropriately sized microcentrifuge tube “HP3.” 16. Prepare 0.1 N HP3 using the calculated reagent volumes. To mix, invert the tube several times. Set aside the 0.1 N HP3 until ready to add it to the NWP plate after the LNW1 washes (see Subheading 3.4, step 31).

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17. Label a new 96-well plate “NLP” for Normalization Library Plate (see Notes 11 and 12). 18. To prepare the NLP plate, begin by inverting the LNS2 tube several times. Aliquot 30 μL LNS2 into the appropriate wells of the NLP plate. This can be performed with a repeater pipette, if available. Cover the wells with a Microseal “B” or strip caps to reduce contamination risk and set the plate aside until the NWP plate is ready for transfer to the NLP plate (see Subheading 3.4, step 35). 19. Once the 30-min shaking incubation of the NWP plate is complete, immediately place the NWP midi plate on the magnetic stand. Carefully remove the seal. Let sit for 2 min or until the liquid is clear and all beads have gathered towards the magnet (see Note 39). Beads gather in opposite directions every other row based on where the magnet is located. 20. Using a 100 μL or similar multi-channel pipette, remove and discard any supernatant from each well (see Note 30). 21. Take the plate off of the magnetic stand and set on a flat surface. 22. Aliquot 100 μL LNW1 per sample/control into a reagent reservoir (this includes enough for two washes and includes extra for pipetting error). 23. Using a 100 μL multi-channel pipette, add 45 μL LNW1 into each well of the NWP midi plate. The plate should not be on the magnetic stand. Retain the remaining LNW1 in the reagent reservoir for the second wash. 24. Seal the NWP midi plate with a Microseal “B” using a plate sealer and shake for 5 min at 1800 rpm. 25. Once shaking is complete, immediately place the NWP midi plate on the magnetic stand. Carefully remove the seal. Let sit for 2 min or until the liquid is clear and all beads have gathered toward the magnet (see Note 39). 26. Using a 100 μL multi-channel pipette, remove and discard any supernatant from each well (see Note 30). Remove the plate from the magnetic stand. 27. Perform a second wash with LNW1 (see Subheading 3.4, steps 23–26). 28. Once two washes have been performed, remove the plate from the magnetic stand, seal the NWP midi plate with a Microseal “B” using a plate sealer, and centrifuge for 30 s at 1000 × g. This should draw any leftover liquid to the bottom of the wells. 29. Carefully remove the seal and place the NWP midi plate back on the magnetic stand. Let sit for 2 min or until the liquid is clear and all beads have gathered toward the magnet (see Note 39).

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30. Remove and discard any leftover liquid from each well using a 20 μL multi-channel pipette. Check that no liquid remains at the bottom of the plate (see Note 33). If liquid is still present, individual wells can be targeted using a single-channel pipette. 31. Invert the prepared 0.1 N HP3 tube a few times and remove the NWP midi plate from the magnetic stand. 32. Dispense 32 μL 0.1 N HP3 into each well of the NWP midi plate. Check that each well is holding equal volumes after pipetting. If using a multi-channel pipette, the 0.1 N HP3 can be dispensed into a reagent reservoir and then added to the NWP plate. 33. Seal the NWP midi plate with a Microseal “B” using a plate sealer and shake for 5 min at 1800 rpm. After 5 min has elapsed, check that all beads have been resuspended in each well. If not, the shake step can be repeated, or individual wells can be mixed using a pipette. 34. Place the NWP midi plate on the magnetic stand. Carefully remove the seal. Let sit for 2 min or until the liquid is clear and all beads have gathered towards the magnet (see Note 39). 35. Remove the cover from the NLP plate that has been set aside. Using a multi-channel pipette, remove 30 μL of each sample from the NWP midi plate and transfer to the corresponding wells of the NLP plate (see Note 34). Mix using the pipette. 36. Seal the NLP plate with a Microseal “B” using a plate sealer or cap strips and centrifuge for 30 s at 1000 × g. 37. The expiration of the normalized plate is 30 days. It can be processed immediately to pool the sample libraries (see Subheading 3.5, step 1) or stored in a freezer (-25 to -15 °C) until ready to proceed. 3.5 Library Preparation—Pooling of Sample Libraries

1. Continue processing the room temperature NLP plate or if stored, remove from the freezer and allow to come to room temperature (see Subheading 3.4, step 37). 2. Determine the maximum number of samples that can be sequenced based on the primer set and the flow cell used (see Table 8 and Note 40).

Table 8 Maximum number of samples for each flow cell kit Flow cell

Primer Set A

Primer Set B

Micro flow cell kit

36

12

Standard flow cell kit

96

32

The standard flow cell and micro flow cell kits each allow a separate maximum number of samples depending on the primer set used

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3. If processing a small sample number of samples/controls from the NLP plate, label a single 1.5 mL microcentrifuge as “PNL” for Pooled Normalized Libraries (see Note 12). For a large number of samples/controls, label a single 8-tube strip instead. 4. Vortex and centrifuge the NLP plate for 30 s at 1000 × g to remove any condensation from the seal or caps. 5. Place the NLP plate on the magnetic stand. Carefully remove the seal. Let sit for 2 min or until the liquid is clear and all beads have gathered toward the magnet (see Note 41). Beads gather in opposite directions every other row based on where the magnet is located. 6. Aliquot 5 μL of each sample into the labeled PNL tube/8-tube strip, changing tips between each sample/row. If sequencing a small number of samples, all samples are added to the single 1.5 mL tube using a single-channel pipette. If sequencing a larger number of samples, use a multi-channel pipette to transfer one column of samples from the NLP plate at a time to the 8-tube strip, repeating for each column into the same strip. This will effectively transfer all samples from a single row of the NLP into the corresponding tube of the 8-tube strip. Lastly, combine each tube in the strip into a final 1.5 mL microcentrifuge tube. 7. Vortex the PNL tube and pulse spin. 8. Do not discard the NLP plate. There is enough volume for additional sequencing. Re-seal and place the NLP plate back into the freezer for storage. 9. The expiration of PNL tube/tube-strip is 30 days. It can be processed immediately to denature and dilute the pooled libraries (see Subheading 3.6, step 1) or stored in a freezer (25 to -15 °C) until ready to proceed. 3.6 Denaturation and Dilution of Pooled Libraries and Cartridge Loading for Sequencing

1. Thaw the denaturation/dilution reagents (~90 min for the Reagent Cartridge, but less for HT1 and HSC). Remove the MiSeq FGx™ Reagent Kit from the freezer; wait to thaw the HSC (see Subheading 3.6, step 3). Remove the HT1 box from the underside of the handle of the reagent cartridge and set it aside to thaw at room temperature. Fill an appropriately sized container with room temperature water and place the reagent cartridge in the water for ~90 min to thaw (see Notes 42 and 43). 2. Set the micro-heating system/heat block to 96 °C. This step can take some time depending on the system. Let the system heat while the reagents are thawing. 3. During the remaining ~15 min needed to thaw the reagent cartridge, remove the HSC (and PNL tube/tube strip, if stored

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from Subheading 3.5, step 9) from the freezer and allow to thaw to room temperature. 4. Before continuing, ensure that all reagents are thawed and there are no ice chunks visible within the reagent cartridge. Once completely thawed, remove the cartridge from the water bath and tap it on the bench to remove water droplets from the surface of the cartridge. Make sure that the base is dry and that no water has pooled on the top of the cartridge. 5. If a benchtop cooler or ice bucket is not available, prepare an ice water bath at this time by mixing 1 part nuclease-free water and 3 parts ice. 6. Label a 1.5 mL microcentrifuge tube as “HSC” for Human Sequencing Control. 7. Vortex and pulse spin the HSC tube from the kit. Add 2 μL HSC, 2 μL HP3, and 36 μL nuclease-free water to the newly labeled HSC tube (see Note 44). Vortex the prepared HSC tube and centrifuge briefly. Let stand at room temperature for 5 min. 8. While the HSC is incubating, label a 1.5 mL microcentrifuge tube as “DNL” for Denatured Normalized Libraries and transfer 591 μL HT1 (hybridization buffer) to the DNL tube. 9. Vortex and pulse spin the PNL tube to remove liquid from the cap. Transfer 7 μL of the PNL to the DNL tube. Flush the tip to mix. Do not discard the PNL tube. There is enough volume left for additional sequencing. Place the PNL tube back in the freezer for storage. 10. After the HSC is done incubating, transfer 4 μL to the DNL tube. Flush the tip to mix and pulse spin. Incubate for 2 min on the preheated micro-heating system set at 96 °C (see Note 45). 11. After incubation, invert the DNL tube several times and immediately place it into the benchtop cooler, ice bucket, or prepared water bath to snap-cool (see Note 46). Incubate for 5 min. 12. During the snap-cool, prepare the reagent cartridge for loading. Invert the cartridge gently 10 times in order to mix the reagents within. Tap the cartridge on a counter gently to remove bubbles and water from the outside of the cartridge. Inspect wells 1, 2, and 4 to make sure they are thoroughly mixed. Using an empty 1000 μL pipette tip, pierce the foil of the well labeled 17. This is to prevent sample loss during the addition of the denatured libraries. Well 17 is highlighted in a deep orange color and is labeled “Load Samples.” 13. After the snap-cool incubation, remove all contents from the bottom of the tube with a pipette set to 600 μL (see Note 47). Slowly (to avoid air bubbles) load all contents into the reagent

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cartridge in the marked well “17”. Once loaded, gently tap the cartridge to ensure all liquid is at the bottom. 14. Immediately proceed to instrument setup and loading (see Subheading 3.7, step 1). 3.7 Instrument Setup and Performing a Run

1. Login to the ForenSeq™ Universal Analysis Software (UAS) and click “Create New Run”. The next screen prompts to choose “Standard” or “Micro”. Choose the appropriate option and click “BEGIN”. 2. Type in the Run Name in the white box under “Run” on the left side of the screen (see Note 12). A run description can be added in the box under “Description” in the middle of the screen. This is not required to save the run. Choose “Forensic Application” from the drop-down menu in the box under “Application” on the right side of the screen. 3. Add samples to the run manually (see Subheading 3.7, step 4), using a tab-delimited (.txt) import file (see Subheading 3.7, steps 5–15), or to an existing run file (see Subheading 3.7, step 16). 4. To manually add samples, click the “ADD NEW SAMPLES” button in blue under the “Name” box. Fill in the “Sample Name” and “Project Name” fields. A “Sample Description” may be chosen but is not required. Choose the appropriate “i7 Index”, “i5 Index”, “Sample Type”, and “Primer Mix” from the respective drop-down menus. Once complete, click the “+ ADD NEW SAMPLE” blue box at the right side of the screen. Complete this action for each sample (see Note 48). Click the blue “Save Run” button at the bottom of the screen. Continue to prepare the instrument for the sequencing run (see Subheading 3.7, step 17). 5. To import a pre-filled samples file, begin by creating a tab delimited (.txt) file using an application such as Microsoft® Excel® (Excel). In the first row, enter the following row headers exactly in the specified cell: “SampleName” (A1); “Project” (B1); “i7Index” (C1); “i5Index” (D1); “SampleType” (E1); “SampleDescription” (F1); and “MixType” (G1) (see Fig. 2; Notes 49 and 50). 6. In column A, type the user-defined sample/control names. 7. In column B, type the user-defined same project name for each sample/control. 8. In column C, type the appropriate i7 Index for each sample/ control, choosing from: “R701”, “R702”, “R703”, “R704”, “R705”, “R706”, “R707”, “R708”, “R709”, “R710”, “R711”, or “R712” (see Note 50).

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Fig. 2 UAS sample file. Displayed is an example of a pre-filled sample file for upload into the UAS software

9. In column D, type the appropriate i5 Index for each sample/ control, choosing from: “A501”, “A502”, “A503”, “A504”, “A505”, “A506”, “A507”, or “A508” (see Note 50). 10. In column E, type the appropriate sample type for each sample/control, choosing from: “Positive Amplification Control”, “Negative Amplification Control”, “Sample”, or “Reagent Blank” (see Note 50). 11. In column F, type the user-defined sample descriptions. These are not required. 12. In column G, type the appropriate primer mix for each sample/ control, choosing from “A” or “B” (see Note 50). 13. After completing the appropriate information for each sample, save the Excel file as a tab-delimited (*.txt) file for upload. 14. To import the file, return to the UAS software and click “IMPORT SAMPLES” directly under the “Name” box. Either drag the .txt file into the box labeled “DROP FILES TO UPLOAD”, or click on the box, navigate to the appropriate .txt file, and click “Open”. 15. Review the information and edit any errors. Click the blue “Save Run” button at the bottom of the screen. Continue to prepare the instrument for the sequencing run (see Subheading 3.7, step 17). 16. To add to an existing sample run, click the “Add Existing Samples” button and type in the sample name to add or the project name of the previous run to find the sample. Fill in the remaining information as described for manual input (see Subheading 3.7, step 4). Click the blue “Save Run” button at the bottom of the screen. Continue to prepare the instrument for the sequencing run (see Subheading 3.7, step 17). 17. Select “Sequence” from the home screen on the MiSeq FGx. Next, select “Forensic Genomics”. The next screen requires

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the correct run folder to be chosen. Click the arrow and a list of loaded runs are available (see Note 51). Choose the correct run file and click “Next”, which initiates a sequence of prompts explained below. 18. Remove the flow cell from the liquid storage container with forceps or drain some of the liquid and manually remove it wearing a glove. Rinse the flow cell with nuclease-free water to remove the storage buffer salts. Dry the flow cell using a lintfree wipe. Make sure the entire flow cell is dry. This may require very careful drying between the glass and plastic areas (see Note 52). Once it is dried, an alcohol wipe can be used to clean the glass of the flow cell. Inspect the flow cell to ensure there is no remaining lint or streaks on the surface. If the gasket has moved, gently push it back into place. 19. Load the flow cell by following the software prompts. Open the flow cell compartment and gently push the white flow cell latch release button, which opens the flow cell latch. The flow cell should be at the top. The notched side of the flow cell should be at the upper right. Close the compartment door. An audible click occurs when the latch is secure. Click “Next”. 20. Load the PR2 bottle following the software prompts. Open the door to the reagent compartment under the screen. Lift up the sipper handle. Remove any bottles currently on the instrument in the PR2 bottle location and replace with a fresh bottle of PR2: invert the new PR2 bottle to mix, remove the lid, and insert. If needed, empty the waste bottle into a biohazard bin and place it back into the instrument. Pull the handle to bring the sipper back down. Click “Next”. 21. Load the loaded reagent cartridge following the software prompts. Lower the reagent chiller door and insert the loaded cartridge. Slide the cartridge on the bottom of the tray until it stops. The handle of the cartridge should be pointed outwards. Close both the reagent chiller door and the reagent compartment door. Click “Next”. 22. The next screen asks to confirm the run name and type of run that will be performed. Check this information and click “Next” if everything is correct. The next screen displays a list of run parameters and performs a run pre-check. Once all parameters have been tested and are in working order, the “Start Run” button is available. Click “Start Run” to begin. 23. Basic run parameters can be monitored during the run from the “Sequencing” screen, including run progress, fluorescence intensity, quality information, as well as current actions and temperatures of the instrument and components. The run can also be stopped and paused from this window [14]. 24. After a run is completed, a post-wash option is available on the screen. Complete a wash immediately after the sequencing is

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complete for the best maintenance of the instrument (see Subheading 3.8, step 1). 3.8 Perform a PostRun Wash

1. After a run is completed, leave the used flow cell in the instrument for the wash run, which is required to complete the wash. 2. Prepare a 1:30 dilution of 6% sodium hypochlorite. Vortex to mix. In a MiSeq wash tube, further dilute the 1:30 to prepare 1000 μL of 0.01% sodium hypochlorite solution. Pipette to mix using a 1000 μL pipette. 3. To load the wash tray, use a large volume repeater pipette to add 6 mL nuclease-free water to each well, except to well 17 (Sample Well). Insert the prepared MiSeq wash tube with 0.01% sodium hypochlorite into well 17 of the wash tray. The tube should be flush with the lip of the well in the tray. 4. Fill an empty PR2 bottle with 350 μL nuclease-free water. 5. Return to the instrument. Click “Start Wash” (see Note 53). Wait a few seconds before opening any doors. The instrument raises the sippers in the used cartridge. 6. Wait until the sippers are fully raised (see Note 54) and replace the used run cartridge with the prepared wash cartridge (see Subheading 3.8, step 3) by opening both the reagent compartment and reagent chiller doors. Close the chiller door after the wash cartridge is completely inserted. Lift up the handle to the sippers on the right side. The wash bottle will replace the leftover PR2 reagent bottle from the run. Remove the previous PR2 bottle and replace it with the wash bottle (see Subheading 3.8, step 4). Empty the waste bottle into a biohazard bin and place it back into the instrument. Lower the sipper handle back down and close all doors. 7. Click “Next” and allow the wash to complete. It lasts about 30 min. 8. Once the wash run is complete, leave all consumables within the instrument, including the wash containers, until the next run is performed. 9. During storage, the wash tray should always be cleaned and turned upside down to decrease the chance of mold growth in the tray.

4

Notes 1. DNA concentration must be known for purified DNA before beginning this process. Quantify the amount of total human DNA using validated qPCR or fluorometric-based assays. 2. The size of tip depends on the brand of pipettes utilized; ensure that the appropriately sized tips are used. It is also best to match

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up the brands of pipette tips and pipettes. Using unmatched tips can cause changes in the volume pipetted/aspirated. 3. Verogen recommends Bio-Rad brand. 4. Only needed for FTA® Card processing. 5. Examples include QuickExtract DNA Extraction Solution or SwabSolution Kit. 6. Normalization reagents are hazardous. Both formamide and 2-mercaptoethanol are used. Handle with caution and dispose of properly. 7. Author uses a ThermoFisher MicroAmp™ Splash-Free 96-Well Base. 8. Author uses a magnetic stand for a 96-well plate. 9. The laboratory can purchase a unit that also is able to heat samples at the same time. Manufacturers recommend BioShake iQ or BioShake XP. 10. Make sure to allow sufficient time before beginning to allow all contents to thaw and resolubilize. This is especially important for the SPB and LNB1 tubes and may affect the binding of DNA to the beads if thawing is incomplete. For LNA1, reagents may crystallize during storage. Vortex thoroughly and check that there are no crystals present. If present, let thaw further and vortex again. Crystals can be best seen if the tube is held up to a light. 11. If processing one column of samples (eight, including all controls), this step can be performed using an 8-tube strip and accompanying 8-cap strip. Ensure that the laboratory has a pulse spinner or microcentrifuge that can fit 8-tube strips. 12. Follow the labeling/naming system for the laboratory. An example labeling system can include the plate/tube type (or analyst initials) and process date. For example, “FSP_011022,” using a two-digit month, two-digit date, and two-digit year. For storage and organization purposes, write the expiration date of the plate, if applicable. 13. If the sample concentration is 8 samples, in which case the master mix should be equally distributed into an 8-tube strip and dispensed into each column of the 96-well amplification plate. Author recommends utilizing a new pipette tip for each well to ensure an even distribution. 18. Flush the tip by pipetting the liquid up and down into the tip, gently, to avoid saturating the barrier. 19. To seal Microseal “A,” push with enough pressure to ensure that the plate has been thoroughly sealed, but not too much pressure that the seal is no longer viable. A clear shadow outline should be visible around each well. If the seal is not flush with each well due to overpressure, gently remove and apply a new seal. 20. These tubes are narrower than the average microcentrifuge tube. It helps to place empty 1.7 or 2.0 mL microcentrifuge tubes into the pulse spinner (caps removed/cut off at the hinge) and place the index tubes inside these empty tubes. 21. The order and number of the indices used depends on the number of samples being sequenced. This should be determined before library prep. 22. The author recommends keeping track of the volume of each index and rotating out the i5 and i7 indices throughout different runs. 23. The FSP2 plate is not secured within the ForenSeq™ Index Plate Fixture. The plate can be placed in a plate holder during pipetting to better stabilize. 24. New caps are used for each assay to ensure that no contamination occurs between index tubes. If sequencing smaller batches, the kit does not contain enough caps and additional caps should be purchased. 25. To avoid air bubbles, pipette PCR2 slowly. For >8 samples, PCR2 can be distributed into an 8-tube strip and dispensed using a multi-channel pipette. For ≤8 samples, use a singlechannel pipette.

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26. During storage, the SPB/LNB1 beads accumulate at the bottom of the tube. During vortexing, it helps to invert the tube multiple times and re-vortex to unclump. 27. When pipetting into the midi plate, the SPB or LNB1 may require additional mixing if the beads have not been in use; this ensures homogeneity. 28. This must be done in order to ensure that all leftover amplification reagents have been removed. 29. This step may last longer than 2 min. If at any point beads are drawn up into the pipette tips inadvertently, dispense the liquid back into the appropriate wells and let sit for approximately 2 min to rebind. 30. Ensure that all liquid has been removed to allow for sufficient washing and sample transfer later in the process. 31. Ensure that there is a space between the tip and the bottom; otherwise, not all liquid is removed. Barely lift up the pipette tip to make sure there is space to draw up all of the liquid. To avoid interacting with the beads, insert the pipette to the opposite side of the beads (plunger down). Touch the tips to the opposite side at the top of the well and slowly move the tips downwards until it reaches the bottom. Once at the bottom, gently straighten the pipette tip. Lift up gently and slowly to aspirate the liquid. 32. Prepare fresh 80% EtOH for every preparation to ensure optimal results. The formula contains overage. 33. It is crucial that no liquid remains. Otherwise, the sample is diluted and less than optimal results may be obtained. 34. Author has observed beads sticking to the side of the pipette tips at this point during this transfer step. It has not been shown to have an impact on the amount of DNA template sequenced, although no published research has been performed at this time to verify. 35. It is crucial that an equal amount of beads are added to each sample so that each sample has an equal representation in the pooled library. Too many beads added can lead to an overabundance of reads generated for that sample, and too little can lead to a decreased read count. 36. It is important to use a pipette tip with a larger bore size so that all beads are transferred. 37. This must be done in order to ensure that all beads are available for the DNA to bind. 38. This is to remove any beads remaining from the purification step from the supernatant. 39. This step tends to clear faster than the purification step and does not always require the full 2 min.

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40. It is recommended to include the sequencing amplification positive and negative controls on each run performed, even if previously sequenced on a different run. If an error has occurred during the sequencing process, and a positive and negative control were not included, the ability to appropriately troubleshoot is lessened, as well as the assistance that Verogen can provide in determining the reason for the problem. 41. This is to remove any beads from the supernatant that are remaining from the normalization step. 42. Denaturation and dilution of the pooled libraries should occur the day of the sequencing run. Once this section is complete, the loaded cartridge must be immediately processed on the instrument to avoid instrument and sequencing errors. 43. The water does not have to be nuclease-free and should not go above the “Fill Line” on the cartridge once it is placed in the bath. Author recommends drawing a line on the container to the appropriate fill line during the first thaw for future uses. Alternatively, the reagent cartridge can be thawed in the refrigerator, which will take ~5–6 h. Once the cartridge is thawed, it can sit on ice or at room temperature for up to 6 h, but it is best practice to load the sample immediately. 44. Do not begin this step until the heating system is up to temperature. Once this step begins, the subsequent steps are timed. 45. Once the sequencing run has started, the HSC tube can be discarded. The manufacturers advise against storage and repeated use of this reagent, as it can lead to less-than-optimal results. 46. Remove the cooler from the freezer right before use. The temperature should be -25 to -15 °C. If removed too early, the temperature may be too warm, and incomplete denaturation may occur. 47. Do not remove any of the contents that may have condensed at the top of the DNL tube during the cooling step. Incomplete denaturation/dilution may have occurred and leads to lessthan-optimal results. 48. Each sample is added to a growing list at the bottom of the screen. If the initial sample setup was done incorrectly, the indices, sample type, and/or primer mix type can be fixed using the appropriate drop-down menus. 49. The author finds this is easiest to set up in Excel. 50. This must be exact, otherwise the instrument does not recognize the content.

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51. The instrument only displays runs that have not been completed. 52. The author has found the easiest way to do this is to work from the underside of the flow cell and slide the edge of the wipe up to the area to dry. Capillary action pulls the liquid out from under the edge onto the wipe. Use extra care around the port gasket. 53. This option becomes available after a sequencing run has been completed. 54. For an indication that the sippers are fully raised, the noise will stop and the action on the screen disappears. References 1. Verogen (2022) ForenSeq DNA Signature Prep Kit reference guide. Available via Verogen. https://verogen.com/wp-content/ uploads/2022/01/forenseq-dna-signatureprep-reference-guide-PCR1-vd2018005-d. pdf. Accessed 31 March 2022 2. Xavier C, Parson W (2017) Evaluation of the Illumina ForenSeq™ DNA Signature Prep Kit—MPS forensic application for the MiSeq FGx™ benchtop sequencer. Forensic Sci Int Genet 28:188–194. https://doi.org/10. 1016/j.fsigen.2017.02.018 3. J€ager A, Alvarez M, Davis C et al (2017) Developmental validation of the MiSeq FGx Forensic Genomics System for targeted next generation sequencing in forensic DNA casework and database laboratories. Forensic Sci Int Genet 28:52–70. https://doi.org/10.1016/j.fsigen. 2017.01.011 4. Moreno L, Galusha M, Just R (2018) A closer look at Verogen’s Forenseq™ DNA Signature Prep Kit autosomal and Y-STR data for streamlined analysis of routine reference samples. Electrophoresis 39:2685–2693. https://doi. org/10.1002/elps.201800087 5. Churchill J, Novroski N, King J et al (2017) Population and performance analyses of four major populations with Illumina’s FGx Forensic Genomics System. Forensic Sci Int Genet 30:81–92. https://doi.org/10.1016/j.fsigen. 2017.06.004 6. Ko¨cher S, Mu¨ller P, Berger B et al (2018) Inter-laboratory validation study of the ForenSeq™ DNA Signature Prep Kit. Forensic Sci Int Genet 36:77–85. https://doi.org/10. 1016/j.fsigen.2018.05.007 7. Gettings K, Kiesler K, Vallone P (2015) Performance of a next generation sequencing SNP assay on degraded DNA. Forensic Sci Int Genet 19:1–9. https://doi.org/10.1016/j. fsigen.2015.04.010

8. Fattorini P, Previdere´ C, Carboni I et al (2017) Performance of the ForenSeq™ DNA Signature Prep Kit on highly degraded samples. Electrophoresis 38:1163–1174. https://doi. org/10.1002/elps.201600290 9. Bornman D, Hester M, Schuetter J et al (2012) Short-read, high-throughput sequencing technology for STR genotyping. Biotech Rapid Dispatches 2012:1–6 10. Sharma V, van der Plaat DA, Liu Y et al (2020) Analyzing degraded DNA and challenging samples using the ForenSeq™ DNA Signature Prep Kit. Sci Justice 60:243–252. https://doi. org/10.1016/j.scijus.2019.11.004 11. Verogen (2021) MiSeq FGx Sequencing System reference guide (VD2018006). Available via Verogen. https://verogen.com/wp-con tent/uploads/2021/02/miseq-fgx-system-ref erence-guide-vd2018006-f.pdf. Accessed 31 Mar 2022 12. Verogen (2017) Library Normalization Wash 1 safety data sheet, revision A. Available via Verogen. https://verogen.com/wp-content/ uploads/2020/06/LP-LNW1-VD202001 7-A.pdf. Accessed 31 Mar 2022 13. Verogen (2021) Introducing the MiSeq FGx Reagent Micro Kit for forensics technical note (VD2018016). Available via Verogen. https:// verogen.com/wp-content/ uploads/2021/02/introducing-miseq-fgxreagent-micro-kit-technical-note-vd2018016b.pdf. Accessed 31 Mar 2022 14. Verogen (2018) ForenSeq™ Universal Analysis Software guide. Available via Verogen. h t t p s : // v e r o g e n . c o m / w p - c o n t e n t / uploads/2018/08/ForenSeq-Univ-AnalysisSW-Guide-VD2018007-A.pdf. Accessed 31 Mar 2022

INDEX A Agarose gel ..........................................130–147, 342, 360 Alu repeats ............................................................ 149–173 Amplification ...................................................3, 4, 11–13, 15–17, 19, 35, 37, 55, 114, 116, 121, 123–125, 131, 144, 149–159, 161–164, 166–173, 176, 177, 182, 185–187, 189, 191, 195, 200, 204, 207–212, 214, 216–225, 228–237, 241–251, 253–262, 264–280, 286, 301, 303, 311, 332, 334, 340–342, 345–347, 368, 370, 371, 400, 401, 405–410, 420, 423–426 Ancestry SNPs ...................................................... 398, 399 Applied Biosystems ................................ 4, 37–39, 53–80, 149, 150, 152, 168, 170, 175–188, 190, 235, 241–251, 264, 265, 275, 285–305, 367–394 Archived latent fingerprints ................................. 351–356 AutoMate Express™ ....................................36, 39, 53–80 Automation ............................................... 36, 37, 84, 369

B Blood ................................................8, 18, 36, 38–44, 46, 55, 56, 60, 61, 63–65, 67, 75, 80, 84–87, 91, 119–123, 208, 216, 219, 223, 228, 232, 235, 255, 257–259, 261, 368, 370, 372, 373, 403 Bone analysis ................................................................. 332 Bone extraction ....................................... 64, 93–102, 337 Buccal cells.................................. 119–125, 222, 235, 266

C Capillary array ............................................ 265, 267, 286, 289, 291, 292, 301 Capillary electrophoresis (CE) .......................3, 9, 12, 19, 35, 236, 243, 247, 257, 259, 260, 263–280, 285–305, 335, 344, 348, 369, 398 ChargeSwitch® Forensic DNA Purification Kit .... 36, 38, 265, 266, 272, 273 CODIS loci ...........................................11, 208, 253, 254 Combined DNA Index System (CODIS) ..................207, 208, 242, 332, 367, 368, 385, 386, 391, 394 Contamination ..........................................4–9, 14–16, 30, 36, 40, 47–49, 54, 57, 65, 67, 69, 77, 78, 80, 83, 84, 90, 95, 113, 116, 125, 132, 153, 167, 169, 178, 182, 184, 216, 217, 219, 220, 222, 223,

232, 233, 243–245, 248, 250, 254, 262, 279, 333, 346, 382, 394, 406, 410, 415, 424 Controls.................................................... 4, 8–12, 18, 19, 24, 30, 40, 41, 43, 46, 54, 60–65, 67–69, 76, 77, 85, 87–89, 105, 119, 120, 132, 134–136, 139, 140, 144, 145, 152, 153, 156, 157, 159, 160, 166–170, 176, 179, 182, 190–193, 195, 197, 200, 202, 209, 210, 212, 214, 216–225, 228, 230–234, 237, 238, 242, 243, 245–250, 254–262, 267, 268, 270, 278, 294, 297, 332–334, 340–342, 347, 363, 365, 372, 373, 377, 378, 381, 382, 385, 389–391, 394, 400, 402, 404–415, 417, 418, 420, 423, 424, 426 Corneocytes................................................. 351, 359, 360

D Degradation index ............................................... 186, 187 Demineralization............................................93–102, 337 Diamond™ Nucleic Acid Dye (DD) .................. 360–365 Differential extraction .......................................24, 35, 46, 61, 103–116 Direct amplification ............................. 19, 208, 210–212, 214, 216–221, 223–225, 227, 229–235, 237, 254, 255, 257–259, 264, 265, 363, 370, 371 DNA ....................................... 3, 23, 35, 53, 83, 93, 103, 120, 129, 149, 175, 189, 207, 227, 241, 253, 264, 285, 307, 331, 351, 359, 367, 397 DNA amplification ..................................... 207–224, 227, 231, 296, 336, 338–340 DNA analysis ...........................3–19, 24, 35, 38, 93, 120, 175, 207, 244, 254, 331–348, 351–356, 367, 368 DNA degradation ....................................... 101, 144, 189 DNA extraction...............................................3, 9, 17, 31, 35–50, 53–80, 83–91, 93, 94, 101, 104, 106, 120, 263–280, 337, 340, 352, 353, 423 DNA IQ™ System.............................. 36–38, 40–43, 105 DNA migration .................................................... 130, 145 DNA polymerase ........................................ 150, 152, 176, 190, 198, 242, 254, 257, 334, 370 DNA purification ................................... 29, 83, 103–116, 119–125, 273 DNA quantification/quantitation ..................... 9–11, 19, 31, 37, 42, 43, 45, 65, 72, 175–188, 236 DNA typing................................................................... 348

Catherine Cupples Connon (ed.), Forensic DNA Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 2685, https://doi.org/10.1007/978-1-0716-3295-6, © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023

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430 Index E

L

Epithelial cells.................................................31, 103, 124 Ethanol precipitation ................................................23–32 Extraction ..................................................4, 9–13, 17–19, 25, 26, 30, 35–44, 46, 47, 49, 54–65, 67, 68, 71–80, 83, 85–91, 93–102, 105–108, 110, 111, 114, 124, 132, 135, 140, 141, 147, 173, 191, 222, 236, 246, 249, 250, 261, 264, 267, 268, 278, 333, 335–337, 339, 346, 355, 356, 368, 370, 393, 403, 405, 408

Latent DNA ......................................................... 359–365 Likelihood ratio (LR) ......................................... 307–311, 316–323, 325, 327, 385–387 Locus .............. 190, 208, 242, 308, 309, 312, 315, 316, 320, 321, 326, 327, 331, 372, 385, 387, 390, 398 Low volume amplification ..................264, 265, 267, 272 LRmix Studio ............................................. 307–309, 311, 312, 314, 315, 317–319, 321–324, 326, 327

F Fast PCR............................................................... 263–280 FBI DISC ...................................................................... 368 ForenSeq™ DNA Signature Prep Kit................ 397, 400, 403, 404 Forensic biology ...............................................18, 95, 351 Forensic DNA ..................................3–19, 24, 35, 36, 38, 39, 53–80, 207, 244, 245, 332, 351–356, 367 Forensic DNA analysis ..................................3–19, 24, 35, 38, 207, 244, 351–356, 367 Forensic DNA sequencing................................... 397–427 Forensic Science ........................... 95, 105, 211, 212, 362 Fragment analysis ....................................... 231–233, 235, 274, 285, 286, 295 FTA® Cards .......................................... 19, 119–125, 223, 235, 400, 403, 404, 407, 408, 423 FTA® Indicating Cards .......................120, 121, 223, 235

G

M MagAttract Suspension G.........................................84, 85 Massively parallel sequencing (MPS) ................. 266, 270, 272, 273, 397 Microcon® centrifugal filter purification .................23–32 Microscopy ......................................................31, 32, 112, 333, 346, 360–362, 364 MiSeq FGx .................................401, 404, 405, 417, 420 Mitochondrial DNA (mtDNA)....................... 5, 331–348 Mixture interpretation .................................................. 227 Molecular sieve .............................................................. 130 Multiplex ........................... 208, 242, 253, 264, 266, 270

N Next generation sequencing (NGS) ........................5, 405 NGM SElect™ Express ......................368, 371–373, 376 Normalized extraction ........................................ 264, 265, 267–270, 272, 278

O ®

GelRed Nucleic Acid Stain ....................... 131, 133, 143 GeneMarker™ HID ..................372, 385, 389, 391, 392 3500 Genetic Analyzer ........................................ 285–305 3500xL Genetic Analyzer ............................................. 285 Genetic Analyzer ........................................ 265–267, 274, 276, 277, 280, 285 GlobalFiler™.............................................. 241–251, 265, 269, 271, 274, 368 GlobalFiler™ Express (GFE) ............................. 244, 368, 370–373, 376, 389, 390, 393, 394

H Hair analysis................................................................... 332

I Investigator 24plex GO!............................................... 261 Investigator 24plex QS ........................................ 253–262

K KAPA2G™ Multiplex Mix ......................... 264, 266, 270

Organic extraction ..................................... 23–32, 35, 36, 106–109, 113, 336–339

P Paramagnetic resin ....................................................36–38 Personal protective equipment (PPE) ................. 4, 5, 18, 30, 40, 84, 95, 122, 143, 178, 222, 229, 245, 255, 267, 352 Phenol......................................................... 23, 25, 28, 95, 97, 113, 114, 333, 337, 339 Phenotypic SNPs........................................................... 399 Polymerase chain reaction (PCR) ...................... 5, 10, 11, 13, 15–17, 19, 23–25, 28, 35, 37, 47, 55, 59, 65, 84, 104, 106, 107, 113, 114, 116, 129, 144, 149, 151, 152, 157, 159, 171, 176–180, 185, 189, 190, 193–195, 198–200, 203, 204, 207, 210, 212, 214, 216–224, 227–236, 241–251, 253–266, 268–272, 274, 278, 286, 332–334, 340, 342, 344, 347, 370, 371, 403, 406–408 PowerPlex® Fusion .................... 207–224, 269, 271, 274

FORENSIC DNA ANALYSIS: METHODS PowerPlex® Y23 .................................................. 227, 228, 230–234, 269, 271, 274 PowerUp™ SYBR® Green Master Mix .............. 149–173 Precautions ....................................................4–12, 24, 40, 105, 121, 143, 245, 250, 267 PrepFiler™ ................................................................53–80 PrepFiler™ BTA............................. 53, 58–60, 63, 70, 76 PrepFiler Express™...............................56, 57, 59, 67, 68 PrepFiler Express™ BTA ..........................................53, 59 Probabilistic modeling .................................................. 308 Probability of drop-in (pDI) ........................................ 309 Probability of drop-out (pDO) .......................... 309–311, 317, 319–321, 327 Promega DNA IQ™ System............................... 103–116

Q QIAamp DNA Blood Mini Kit ...................38, 40–42, 46 QIAamp DNA Investigator Kit........38, 40–43, 353, 354 QIAamp DNA Mini Kit ..............................38, 40–42, 46 QIAGEN BioSprint® 96 (BioSprint® workstation) ..............................83–91 Quality assurance ..................................... 4, 9, 17–19, 24, 46, 129, 149, 264, 368 Quality control ............................................ 6, 18, 24, 244 Quality sensors (QS) markers.............................. 253, 254 Quantification .......................................... 3, 9, 10, 13, 19, 31, 37, 129, 130, 140, 144, 150–152, 164, 167, 168, 172, 175–188, 191–193, 195–197, 203, 236, 255, 261, 265, 277, 278, 400, 401 Quantifiler Trio .................................................... 176, 180 Quantitation ...........................................4, 10, 11, 19, 42, 43, 45, 55, 65, 72, 77, 78, 91, 109, 110, 156, 175–188, 191, 195, 204, 250, 269, 272, 370, 407 Quantitative gel electrophoresis .......................... 129–147 Quantitative PCR (qPCR)................................10, 11, 37, 116, 131, 149–173, 176–178, 189, 193, 420

R Rapid DNA....................................................... 4, 367–394 RapidHIT™ ......................................................... 367–394 RapidINTEL™ ................. 368, 370, 373, 376, 389, 393

AND

PROTOCOLS Index 431

RapidLINK™............................................. 368, 370, 372, 373, 377, 379, 383–389, 391, 393 Rapid STR analysis ........................................................ 369 7500 Real-Time PCR System ............................ 149, 152, 154, 176, 190 Real-time qPCR ............................................................ 190

S Saliva ....................................................... 5, 40–43, 61, 67, 119–125, 368, 370, 373, 403 SDS software .............................................. 152, 155–157, 161, 166, 167, 170, 171 Sexual assault evidence.................................................. 190 Short tandem repeat (STR) .................................. 3, 5, 12, 37, 116, 124, 125, 149, 189, 207, 208, 227, 242, 253–255, 258, 259, 262, 312, 331, 351–356, 363, 368, 369, 372, 398, 399, 401 Silica .........................36–39, 47–49, 55, 56, 85, 352, 355 Single nucleotide polymorphism (SNP) ...................... 399 SNP analysis................................................................... 397 Spermatozoa......................................................... 103, 104 Standard curve.............................10, 151, 155, 156, 158, 162–164, 167, 171, 172, 177, 178, 181–183, 185, 186, 191, 192, 195, 197, 200, 201, 203, 204 STR amplification..................................... 11–12, 37, 116, 120–124, 165, 173, 263–280 STR profiles ..........................................11, 12, 19, 35, 55, 124, 125, 149, 165, 204, 253, 264, 265, 267, 274, 276–278, 280, 301, 308, 331, 352, 368–372 STR sequencing ................................................... 242, 401 SYBR® Green I dye......................................150–152, 166

T Thermo Fisher Scientific...................................57, 58, 66, 69–71, 74, 141, 146, 160, 161, 164, 368, 374, 375, 378–380, 384, 386, 389 Touch DNA....................... 40–43, 61, 80, 351, 352, 368

Y Yield gel ....................................................... 130, 139, 141 Y-STR ................................ 152, 208, 227, 242, 253, 399