Performance of the Early Access AmpliSeq™ Mitochondrial Panel with degraded DNA samples using the Ion Torrent™ platform

Abstract

The Early Access AmpliSeq™ Mitochondrial Panel amplifies whole mitochondrial genomes for phylogenetic and kinship identifications, using Ion Torrent™ technology. There is currently limited information on its performance with degraded DNA, a common occurrence in forensic samples. This study evaluated the performance of the Panel with DNA samples degraded in vitro, to mimic conditions commonly found in forensic investigations. Purified DNA from five individuals was heat-treated at five time points each (125°C for 0, 30, 60, 120, and 240 min; total n = 25). The quality of DNA was assessed via a real-time DNA assay of genomic DNA and prepared for massively parallel sequencing on the Ion Torrent™ platform. Mitochondrial sequences were obtained for all samples and had an amplicon coverage averaging between 66X to 2803X. Most amplicons (157/162) displayed high coverages (452 ± 333X), while reads with less than 100X coverage were recorded in five amplicons only (90 ± 5X). Amplicon coverage was decreased with prolonged heating. At 72% strand balance, reads were well balanced between forward and reverse strands. Using a coverage threshold of ten reads per SNP, complete sequences were recovered in all samples and resolved kinship and, haplogroup relations. Additionally, the HV1 and HV2 regions of the reference and 240-min heat-treated samples (n = 10) were Sanger-sequenced for concordance. Overall, this study demonstrates the efficacy of a novel forensic Panel that recovers high quality mitochondrial sequences from degraded DNA samples.

An 18 Multi‐InDels panel for analysis of highly degraded forensic biological samples

“…On the other hand, the application of new genetic markers to the genotyping of highly degraded samples has made great progress. Single nucleotide polymorphisms (SNPs) are being studied and applied for identifying degraded DNA due to the low mutation rate and short amplicon [8] and lots of interesting work have been reported [9][10][11][12][13]. Rachel et al [14] focus on investigating highly degraded forensic samples, provided a 27 identity-testing SNPs panel with amplicon lengths smaller than 50bp.…”

From Forensics to Clinical Research: Expanding the Variant Calling Pipeline for the Precision ID mtDNA Whole Genome Panel

“…Recently, a novel approach to mtDNA NGS through whole genome sequencing (WGS) has been developed with the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific, USA) [ 25 ]. This library preparation kit, with 162 amplicons that target the whole mtDNA, is mostly used for forensic samples, achieving reliable results in often degraded samples [ 26 , 27 , 28 , 29 ] and with low DNA input (usually 0.1 ng of genomic DNA, but 6.25 pg [ 30 ] and, very recently, 0.6 pg [ 31 ] have been reported). Despite its most common use in forensic sciences, it has also been used in a range of other applications, from worldwide lineage studies [ 32 ], to rare mtDNA differences in monozygotic twins [ 33 ].…”

Full mtDNA genome sequencing of Brazilian admixed populations: A forensic-focused evaluation of a MPS application as an alternative to Sanger sequencing methods

“…Hyper-variable regions I and II (HV-I and HV-II, respectively) encompass most of the individual variation among samples, even those belonging to the same haplotype, and as such are considered the most informative region of the mtDNA genome [4]. However, its complete sequence comprises over 16k base pairs, and ˜75% of total reported polymorphic variants are observed in nucleotide positions located in the coding region, usually not included in forensic mtDNA examination based on limitations inherent to SS applications [5,6].…”

“…It was designed to generate small-sized amplicons and successfully allow processing of highly degraded, low-input and other challenging forensic samples. Validation studies show that robust, sensitive, efficient and reliable typing of forensic samples can be accomplished with different versions of this product, allowing its use in forensic individual identification or parentage testing [5,[15][16][17].…”

“…Adoption of proposed analytical alternatives in current forensic casework also demands protocol validation with real evidence obtained from crime scenes. Several studies [5,[15][16][17]26] have already evaluated present commercial solution (as well as posterior versions of the same product) efficiency in mock or real forensic cases with remarkable success. Some particular issues intrinsically linked to the very nature of biological evidence in criminal DNA analysis have also been addressed by several research groups regarding mtDNA evaluation, as mixture occurrence or samples presenting highly degraded DNA or DNA template low-copy numbers [5,[15][16][17]26].…”