The QIAGEN 140-locus single-nucleotide polymorphism (SNP) panel for forensic identification using massively parallel sequencing (MPS): an evaluation and a direct-to-PCR trial
Massively parallel sequencing (MPS) of identity informative single-nucleotide polymorphisms (IISNPs) enables hundreds of forensically relevant markers to be analysed simultaneously. Generating DNA sequence data enables more detailed analysis including identification of sequence variations between individuals. The GeneRead DNAseq 140 IISNP MPS panel (QIAGEN) has been evaluated on both the MiSeq (Illumina) and Ion PGM™ (Applied Biosystems) MPS platforms using the GeneRead DNAseq Targeted Panels V2 library preparation workflow (QIAGEN). The aims of this study were to (1) determine if the GeneRead DNAseq panel is effective for identity testing by assessing deviation from Hardy-Weinberg (HWE) and pairwise linkage equilibrium (LE); (2) sequence samples with the GeneRead DNAseq panel on the Ion PGM™ using the QIAGEN workflow and assess specificity, sensitivity and accuracy; (3) assess the efficacy of adding biological samples directly to the GeneRead DNAseq PCR, without prior DNA extraction; and (4) assess the effect of varying coverage and allele frequency thresholds on genotype concordance. Analyses of the 140 SNPs for HWE and LE using Fisher’s exact tests and the sequential Bonferroni correction revealed that one SNP was out of HWE in the Japanese population and five SNP combinations were commonly out of LE in 13 of 14 populations. The panel was sensitive down to 0.3125 ng of DNA input. A direct-to-PCR approach (without DNA extraction) produced highly concordant genotypes. The setting of appropriate allele frequency thresholds is more effective for reducing erroneous genotypes than coverage thresholds.
KeywordsMPS Forensic Identity SNP DNAseq panel Direct-to-PCR
The authors were supported by QIAGEN through their financial and technical contributions to this project.
Compliance with ethical standards
Conflict of interest
This study was partially funded by QIAGEN. N. Jones and I. Petermann are employed by QIAGEN.
Research involving human participants
Ethics approval was obtained from the University of Canberra Human Research Ethics Committee (Project Number CEHR 14-70), in accordance with the Helsinki Declaration.
Research involving animal participants
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The Chimpanzee blood sample was donated from the sample collection of the Australian Federal Police (AFP).
Informed consent was obtained from all individual participants included in the study.
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