Highlights
- •Pooled PCR amplicons from five forensic SNaPshot assays were applied to Ion Torrent.
- •Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips.
- •136 unique SNPs were accurately genotyped from 0.1 to 0.3 ng template DNA.
- •High concordance (97%) was observed between Ion Torrent and SNaPshot genotypes.
- •Sanger sequencing confirmed a further 1.5% of genotypes discordant with SNaPshot.
Abstract
Forensic phenotyping can provide useful intelligence regarding the biogeographical
ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary
sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and
EVCs is performed most commonly using SNaPshot®, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is
limited to 30–40 SNPs. Next generation sequencing (NGS) offers the potential to genotype
hundreds to thousands of SNPs from multiple samples in a single experimental run.
The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three
different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR
amplicons containing 136 unique SNPs were sequenced using Life Technologies’ Ion Torrent™
PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template
amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot
across all three template amounts. Of those genotypes discordant with SNaPshot, six
Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the
three template amounts. Seven SNPs (1.7%) were either discordant between template
amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative
control, and, allele coverage variation for heterozygous genotypes highlights the
need to establish a threshold for background levels of sequence output and heterozygous
balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable
potential for use in forensic DNA analyses as a low to medium throughput NGS platform
using established SNaPshot assays.
Keywords
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Article info
Publication history
Published online: August 30, 2014
Accepted:
August 25,
2014
Received in revised form:
August 14,
2014
Received:
July 2,
2014
Identification
Copyright
© 2014 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.
