Abstract: This study evaluated the ability to produce FORensic Capture Enrichment (FORCE) genotypes using amplicon-based and capture-based enrichment assays. The FORCE panel is a standardized set of single nucleotide polymorphism (SNP) markers developed for forensic applications. Twelve DNA samples were prepared and distributed to the laboratories for testing: five control DNA samples, a dilution series ranging from 10ng to 0.03ng, two degraded DNA samples with 200bp and 150bp average fragment lengths, and one inhibited sample spiked with humic acid. Fifteen laboratories from three different continents participated in this study, choosing from one of four manufacturer-developed enrichment assays to complete the experiments, setting their own parameters for sequencing and other user-defined steps to accommodate their own preferences and expertise. A total of eighteen methods were evaluated, as three laboratories performed two methods. The results showed that all four assays were successful in producing full FORCE SNP genotypes from high quality samples. However, significant differences between and within assays and methods were observed. Read count variability and enrichment type led to significant differences in call rate. Robust SNP recovery was observed across all assays at 0.3ng DNA input, with an amplicon-based assay producing high SNP call rates at 0.03ng DNA input. Capture and single primer extension assays produced consistently high SNP call rates from degraded samples with 150-200bp fragments. Future research to optimize laboratory parameters may reduce the variation in SNP data, so that labs may equitably adopt SNP methods to make use of these powerful forensic markers.
Authors:
Andreas Tillmara,b,1, Kimberly Sturk-Andreaggic,d,1, Adam Staadiga,b, Christina Amorye, David Ballardf, Erik F. Bergsethg, Claus Børstingh, Selena Cisanai, Michael Coblej, Lucinda Davenportf, María de la Puentek, Katherine B. Gettingsl, Erin M. Gordenm, Kelly Grisedalen, Jennifer Daniels-Higginbothamc,d,2, Rachel Houstono, Sheree Hugheso, Reet Järvingp, Kevin M. Kieslerl, Jonathan L. Kingj, Maria Victoria Lareuk, Odile Loreilleq, Dennis McNevinn,r, Melissa K. Muenzlerj, Walther Parsone,s, Vania Pereirah, Christopher Phillipsf,k, Jorge Ruiz-Ramírezk, Maarja Sadamp, Jennifer Snedekero, Carolyn R. Steffenl, Monika Stoljarova-Bibbp, Peter M. Vallonel, Jodie Wardn,r, Jessica Watsonn,r, Catarina Xaviere, Daniel Klinga,g,t, Charla Marshallc,d,s,u,2
aDepartment of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Sweden
bDepartment of Biomedical and Clinical Sciences, Faculty of Medicine and Health Sciences, Linköping University, Sweden
cArmed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL), Dover Air Force Base, USA
dSNA International, LLC, Alexandria, USA
eInstitute of Legal Medicine, Medical University of Innsbruck; Innsbruck, Austria
fKing’s Forensics, King’s College London, United Kingdom
gDepartment of Forensic Sciences, Oslo University Hospital, Norway
hDepartment of Forensic Medicine, University of Copenhagen, Denmark
iCentro Regionale Antidoping, Turin, Italy
jCenter for Human Identification University of North Texas Health Fort Worth, USA
kInstitute of Forensic Sciences, University of Santiago de Compostela, Spain
lApplied Genetics Group, National Institute of Standards and Technology, USA
mSignature Science, LLC, USA
nNational DNA Program for Unidentified and Missing Persons, Australian Federal Police, Australia
oDepartment of Forensic Science, Sam Houston State University, USA
pEstonian Forensic Science Institute, Estonia
qDNA Support Unit, Federal Bureau of Investigation Laboratory, USA
rCentre for Forensic Science, University of Technology Sydney, Australia
sForensic Science Program, The Pennsylvania State University, USA
tBiostatistics (BIAS), Norwegian University of Life Sciences, Norway
uCarl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, USA
1Department of Biomedical and Clinical Sciences, Faculty of Health Sciences, Linköping University, Linköping, Sweden
2contractor supporting the AFMES-AFDIL