Abstract
Forensic mitochondrial DNA (mtDNA) analysis conducted using next-generation sequencing (NGS), also known as massively parallel sequencing (MPS), as compared to Sanger-type sequencing brings modern advantages, such as deep coverage per base (herein referred to as read depth per base pair (bp)), simultaneous sequencing of multiple samples (libraries) and increased operational efficiencies. This report describes the design and developmental validation, according to forensic quality assurance standards, of end-to-end workflows for two multiplexes, comprised of ForenSeq mtDNA control region and mtDNA whole-genome kits the MiSeq FGxTM instrument and ForenSeq universal analysis software (UAS) 2.0/2.1. Polymerase chain reaction (PCR) enrichment and a tiled amplicon approach target small, overlapping amplicons (60–150 bp and 60–209 bp for the control region and mtGenome, respectively). The system provides convenient access to data files that can be used outside of the UAS if desired. Studies assessed a range of environmental and situational variables, including but not limited to buccal samples, rootless hairs, dental and skeletal remains, concordance of control region typing between the two multiplexes and as compared to orthogonal data, assorted sensitivity studies, two-person DNA mixtures and PCR-based performance testing. Limitations of the system and implementation considerations are discussed. Data indicated that the two mtDNA multiplexes, MiSeq FGx and ForenSeq software, meet or exceed forensic DNA quality assurance (QA) guidelines with robust, reproducible performance on samples of various quantities and qualities.
Highlights
Human mitochondrial haplotype testing is valuable in forensically relevant scenarios [1,2,3,4,5,6,7,8,9]
The 16 challenged samples were processed with the ForenSeq mitochondrial DNA (mtDNA) control region kit and the ForenSeq mtDNA whole-genome kit
A minimum of eight mtDNA control region or mtGenome libraries is recommended to be processed at a time, including positive and negative controls if used, to avoid the introduction of pipetting inaccuracies when preparing master mixes due to small volumes
Summary
Human mitochondrial haplotype testing (mitotyping) is valuable in forensically relevant scenarios [1,2,3,4,5,6,7,8,9]. Forensic mtDNA analyses by Sanger sequencing largely focused on two hypervariable regions (HVI, HVII) within the control region (CR, approximately 1122 bp), so-called as it contains the mitochondrial origin of replication and transcription This small portion of the mitochondrial genome (mtGenome, approximately 16,569 bp) has been most explored due to its noncoding nature and the level of effort required to generate 1–2x sequencing coverage with chain termination chemistry. ForenSeq UAS 2.0/2.1 automatically generates mtDNA variant calls as compared to the revised Cambridge reference sequence (rCRS) and using standardized nomenclature accepted by the global forensic science community [42] This software was designed for forensic use and provides a relatively flat workflow as compared to research tools: it does not require multiple programs to deliver results and includes tools and reports intended to address casework and databasing needs. RReessuullttaanntt ppaaiirreedd--eenndd,, dduuaall--iinnddeexxeedd,, ttiilleedd lliibbrraarriieess aarree awammaprpelli,ifif(iebedd),,FppouurrerinififSieeeddq,,apanrndodtpopocoooollelddediivniintdoteoosnoeenacetuhtbuDebNfeoAfroMrsaMiSmeiSpqelFeqGiFnxGtsoxeqtswueqeonuPceiCnncRgisnagnwdaitnahdnsaaelnypsaailrsyawstiesitphwrFiimtohreeFrnosSreeetqnsSU(esAqetSU1oA,rsSoettohr2e)or itsnhoefartwtsioalerfted-, (sbtr)aFtoegreynStheaqtpprrootomcootledsiveifdfiecsieenatchamDpNliAficsaatmiopnleoifnotovetwrlaopPpCinRgs wamithplsiecopnarsattoe parlliomwercsoemtsp(sleette1c, osevte2ra) gine aantidledresdtruacteesgyunthinatpternodmeodtebsyepffirocdieuncttas,m(cp)litfihceattwioon-PofCoRvearplpapropainchg caamnpflaiccoilnitsattoe caollnofwirmcoamtipolnetoefcvoavreiarangt(es)anthdarterdeusicdees uunndinetrenadperdimbeyrp:rwodhuecntsa, (pcr)itmhert-wbion-dPiCnRg saiptepmrouactahticoannefxaicsitlsitautnedceornafiprmrimatieorninofovnaerpiarnimt(se)r tsheatt(aressteidrieskunudnedrear aprsiemt 1erp: rwimhenr ianpthriims deri-abgirnadmin),gthsietne mthuatavtaiorinanext icsatns ubendreelriaabplyridmeetercitnedonine apmrimpleicrosnest e(axstetenrdisekdufrnodmerthaesectom prainmioenr ipnritmhiesrdsieatg(rsaemt 2)),.tAheltnertnhativearaipanptrocanchbees rcealniambloyrdeeftreecqteudenintlyamprpolidcuocnes aemxtbenigdueidtyfrroemgatrhdeincgomprpiamneior nsepqruimenecressevt e(srestu2s)v. aArlitaenrtnsa.tive approaches can more frequently produce ambiguity regarding primer sequences versus variants
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