Massively parallel sequencing of short tandem repeats—Population data and mixture analysis results for the PowerSeq™ system

Abstract

Current forensic DNA analysis predominantly involves identification of human donors by analysis of short tandem repeats (STRs) using Capillary Electrophoresis (CE). Recent developments in Massively Parallel Sequencing (MPS) technologies offer new possibilities in analysis of STRs since they might overcome some of the limitations of CE analysis. In this study 17 STRs and Amelogenin were sequenced in high coverage using a prototype version of the Promega PowerSeq™ system for 297 population samples from the Netherlands, Nepal, Bhutan and Central African Pygmies. In addition, 45 two-person mixtures with different minor contributions down to 1% were analysed to investigate the performance of this system for mixed samples. Regarding fragment length, complete concordance between the MPS and CE-based data was found, marking the reliability of MPS PowerSeq™ system. As expected, MPS presented a broader allele range and higher power of discrimination and exclusion rate. The high coverage sequencing data were used to determine stutter characteristics for all loci and stutter ratios were compared to CE data. The separation of alleles with the same length but exhibiting different stutter ratios lowers the overall variation in stutter ratio and helps in differentiation of stutters from genuine alleles in mixed samples. All alleles of the minor contributors were detected in the sequence reads even for the 1% contributions, but analysis of mixtures below 5% without prior information of the mixture ratio is complicated by PCR and sequencing artefacts.

Comprehensive diagnostics of acute myeloid leukemia by whole transcriptome RNA sequencing

“…PCR products were purified using Wizard SV Gel and PCR Clean-Up System kit (Promega Corporation). For targeted NGS on cDNA, libraries were prepared from PCR products by ligating barcoded TruSeq adapters (Illumina) using the KAPA Library Preparation kit (KAPA Biosystems) [18]. After pooling the libraries, sequencing was performed on the MiSeq sequencer (Illumina) using v3 sequencing reagents according to the manufacturer's protocol.…”

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

“…As is common practise in forensic genetics, such novel tools undergo detailed evaluation [3,[8][9][10][11][12] and validation [1,13,14] prior to their application in casework. Recent population studies have shown that sequencing of STRs lead to an increased power of discrimination compared to commonly used CE sizing by a) illustrating micro-variant structures and b) identifying sequence variations located in the repeat-(isometric variants; alleles of identical size but different in internal sequence) or flanking region [15][16][17][18][19][20]. Moreover, current MPS-kits provide the option to multiplex various loci simultaneously within one reaction, such as STRs (autosomal, Y-and X-chromosomal), mitochondrial DNA (control region) as well as single nucleotide polymorphisms (SNPs) that provide estimates of identity, phenotypic traits, biogeographical and ancestry information [21][22][23][24].…”

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems,

“…In addition, NGS has potential advantages with challenging samples and complicated familial testing (6,8,9). Therefore, increasing numbers of research groups are dedicating substantial efforts to studying NGS-based STR genotyping (10)(11)(12)(13)(14)(15)(16).…”

“…Bornman et al's (5) study on short-read STR genotyping was an exception. Since 2015, the research community has shifted work to Miseq (7,11,12,14,16,(23)(24)(25)(26)(27) and Ion Torrent NGS machines (9,10,13,15,28) because 454 sequencers are no longer supported by their producer. Typing results for 48 autosomal STR markers (11,12,22,24), 29 Y-STR markers (10,24,25), and 9 X-STR markers (24) have been reported.…”