Integrated massively parallel sequencing of 15 autosomal STRs and Amelogenin using a simplified library preparation approach

Abstract

Massively parallel sequencing (MPS) technologies, also termed as next-generation sequencing (NGS), are becoming increasingly popular in study of short tandem repeats (STR). However, current library preparation methods are usually based on ligation or two-round PCR that requires more steps, making it time-consuming (about 2 days), laborious and expensive. In this study, a 16-plex STR typing system was designed with fusion primer strategy based on the Ion Torrent S5 XL platform which could effectively resolve the above challenges for forensic DNA database-type samples (bloodstains, saliva stains, etc.). The efficiency of this system was tested in 253 Han Chinese participants. The libraries were prepared without DNA isolation and adapter ligation, and the whole process only required approximately 5 h. The proportion of thoroughly genotyped samples in which all the 16 loci were successfully genotyped was 86% (220/256). Of the samples, 99.7% showed 100% concordance between NGS-based STR typing and capillary electrophoresis (CE)-based STR typing. The inconsistency might have been caused by off-ladder alleles and mutations in primer binding sites. Overall, this panel enabled the large-scale genotyping of the DNA samples with controlled quality and quantity because it is a simple, operation-friendly process flow that saves labor, time and costs.

The forensic landscape and the population genetic analyses of Hainan Li based on massively parallel sequencing DNA profiling

“…The large-scale performances and functional tests had been widely validated for the ForenSeq™ DNA Signature Prep Kit, the DNA Primer Set A (DPMA, 152 loci totally: 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 identity informative SNPs) and the DNA Primer Set B (in total 230 loci: DMPA plus 22 phenotypic informative SNPs and 56 biogeographical ancestry SNPs), which also include robustness, reproducibility, species specificity, consistency and sensitivity of detection [40][41][42][43][44][45][46][47]. In addition, the applicability of the DNA Signature Prep Kit had been evaluated and verified on challenging samples, DNA mixture, degradative DNA [48][49][50], formalin-fixed paraffin-embedded tissues [49,51], and remains of skeletons and bones [48,[52][53][54], to extend the applications for forensic sciences [55][56][57][58][59][60][61][62][63][64][65][66][67][68]. Hence, on the basis of the sufficient validations of the stability and homogeneity, more essential data of worldwide populations are needed urgently at present to improve the standard experimental procedures, promote the efficiencies of MPS-based system, and strengthen and unify the contacts between capillary electrophoresis-based (CE-based) and MPS-based data for the basic and extended applications of forensic sciences and others.As the aborigines in Hainan island where was the entrances to East Asia (either the southern entrance from Indo-China peninsula or the northern entrance from Central Asia) [12,[69][70][71], Hainan...…”

High polymorphism detected by massively parallel sequencing of autosomal STRs using old blood samples from a Chinese Han population

“…Then, 93.4–95.6% (3.2–4.1 M) of the length-qualified reads were successfully sorted into different sample libraries and then into different STR libraries. In the last filtering step, 2.5–3.7 M of the previously sorted reads were qualified as reads with complete repeat sequences by identifying a “downstream barcode” 11 . At last 2.4–3.6 M were successfully matched to the pre-constructed reference, and there were no significant data volume deviations among the four sequencing runs.…”

“…Libraries containing 16 STR amplicons (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D2S1338, D19S433, and amelogenin) attached by adaptors were directly amplified from whole blood using the fusion primer pool and blood direct DNA polymerase (SeqType ® R16 Kit, IPE Biotechnology, Beijing, China). The details of the fusion primer have been reported previously 11 .…”

“…PCR was performed in a 10 µL reaction volume as described previously 11 with 1 µL of whole blood as the template. Equal volumes of 192–204 PCR products were pooled (190–202 samples and two 2800 M controls), and then 50 µL of this pool was purified with 1.2 × volume SeqType ® R16 DNA clean-up reagent (IPE Biotechnology, Beijing, China).…”

“…The reference bank in this software was based on Chinese Han population data collected by IPE Biotechnology Co., Ltd. The analysis was performed similar to the pipelines in a previous study 11,24 . Allele (30× to 50×) or locus (80×) coverage cut-offs were set up by assuming a binomial distribution of heterozygous allele coverage, based on absolute amplification balance and no read length effect on sequencing quality.…”