Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™

Abstract

Next generation sequencing (NGS) offers the opportunity to analyse forensic DNA samples and obtain massively parallel coverage of targeted short sequences with the variants they carry. We evaluated the levels of sequence coverage, genotyping precision, sensitivity and mixed DNA patterns of a prototype version of the first commercial forensic NGS kit: the HID-Ion AmpliSeq™ Identity Panel with 169-markers designed for the Ion PGM™ system. Evaluations were made between three laboratories following closely matched Ion PGM™ protocols and a simple validation framework of shared DNA controls. The sequence coverage obtained was extensive for the bulk of SNPs targeted by the HID-Ion AmpliSeq™ Identity Panel. Sensitivity studies showed 90-95% of SNP genotypes could be obtained from 25 to 100pg of input DNA. Genotyping concordance tests included Coriell cell-line control DNA analyses checked against whole-genome sequencing data from 1000 Genomes and Complete Genomics, indicating a very high concordance rate of 99.8%. Discordant genotypes detected in rs1979255, rs1004357, rs938283, rs2032597 and rs2399332 indicate these loci should be excluded from the panel. Therefore, the HID-Ion AmpliSeq™ Identity Panel and Ion PGM™ system provide a sensitive and accurate forensic SNP genotyping assay. However, low-level DNA produced much more varied sequence coverage and in forensic use the Ion PGM™ system will require careful calibration of the total samples loaded per chip to preserve the genotyping reliability seen in routine forensic DNA. Furthermore, assessments of mixed DNA indicate the user's control of sequence analysis parameter settings is necessary to ensure mixtures are detected robustly. Given the sensitivity of Ion PGM™, this aspect of forensic genotyping requires further optimisation before massively parallel sequencing is applied to routine casework.

SNP-Based Fetal DNA Detection in Maternal Serum Using the HID-Ion AmpliSeq™ Identity Panel

“…Only limited information can be gained from DNA profiles of a mixed sample without reference genotypes, particularly regarding the genotype of the minor contributor, and so several indicators have been developed to best assess these samples [5]. Multiple studies have shown that heterozygote balance in a sample from a single heterozygous individual can range from 40%-60% [4,5,7,8]; however, it is not possible to define exact limits for homozygotes and heterozygotes in 1:1 mixtures.…”

“…Multiple studies have shown that heterozygote balance in a sample from a single heterozygous individual can range from 40%-60% [4,5,7,8]; however, it is not possible to define exact limits for homozygotes and heterozygotes in 1:1 mixtures. This is because the balance varies depending on the relative amounts of DNA from each individual and on whether the minor contributor is the homozygote or the heterozygote [5]. However, imbalanced patterns in the heterozygote balance of mixed samples with 10% or less of the DNA from the minor contributor are routinely detected [5].…”

“…This is because the balance varies depending on the relative amounts of DNA from each individual and on whether the minor contributor is the homozygote or the heterozygote [5]. However, imbalanced patterns in the heterozygote balance of mixed samples with 10% or less of the DNA from the minor contributor are routinely detected [5]. Changes in the observed level of heterozygosity in mixtures were also discussed.…”

“…High-throughput nucleotide sequencing identification and has been evaluated for coverage, heterozygote balance, and sensitivity in multiple validation studies [4][5][6][7][8]. This panel can reliably provide genotype information given small amounts of template DNA, as well as allow quantitative analyses.…”

“…Here, we report our findings on fDNA detection in maternal serum samples using the HIDIon AmpliSeq TM Identity Panel. Because of the absence of reference information for fetuses and mothers, interpretation of the results was performed using criteria from previous studies [5,8].…”