Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories.
It offers higher resolution to address most problems of human identification,
greater efficiency and potential ability to interrogate very challenging forensic casework
samples. In this study, a trial set of DNA samples was artificially degraded by progressive
aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample
and control sample 2800 M, to test the performance and reliability of the ForenSeqTM
DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate
tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and
0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the
NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples
showed a very limited number of reads/sample, from 2.9–10.2 folds lower than the
ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was
impossible to assign up to 78.2% of the genotypes in the degraded samples as the software
identified the corresponding loci as “low coverage” ( 50x). Amplification artifacts such
as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded
samples. However, the ForenSeqTM DNA Sequencing kit, on the Illumina MiSeq,
was able to generate data which led to the correct typing of 5.1–44.8% and 10.9–58.7% of
58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed
higher chances to be typed correctly compared to the STRs. This NGS approach showed
very promising results in terms of ability to recover genetic information from heavily degraded
DNA samples for which the conventional PCR/CE approach gave no results. The
frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of
the degraded samples. However, these results suggest that further validation studies and
a definition of interpretation criteria for NGS data are needed before implementation of
this technique in forensic genetics.