Abstract
The application of next generation sequencing (NGS) for the analysis of mitochondrial (mt) DNA, short tandem repeats (STRs), and single nucleotide polymorphism (SNPs) has demonstrated great promise for challenging forensic specimens, such as degraded, limited, and mixed samples. Target enrichment using probe capture rather than PCR amplification offers advantages for analysis of degraded DNA since two intact PCR primer sites in the template DNA molecule are not required. Furthermore, NGS software programs can help remove PCR duplicates to determine initial template copy numbers of a shotgun library. Moreover, the same shotgun library prepared from a limited DNA source can be enriched for mtDNA as well as nuclear markers by hybrid capture with the relevant probe panels. Here, we demonstrate the use of this strategy in the analysis of limited and mock degraded samples using our custom probe capture panels for massively parallel sequencing of the whole mtgenome and 426 SNP markers. We also applied the mtgenome capture panel in a mixed sample and analyzed using both phylogenetic and variant frequency based bioinformatics tools to resolve the minor and major contributors. Finally, the results obtained on individual telogen hairs demonstrate the potential of probe capture NGS analysis for both mtDNA and nuclear SNPs for challenging forensic specimens.
Highlights
Generation sequencing (NGS) has shown great promise in the analysis of DNA from degraded, limited, and mixed samples often encountered in forensic cases [1,2,3]
Next generation sequencing (NGS) analysis of short tandem repeats (STRs) polymorphism has revealed additional sequence polymorphisms within tandem repeats not identified by conventional capillary electrophoresis approaches [9,10]
These results demonstrated the specificity of our custom probe capture NGS method; of the average total of
Summary
Generation sequencing (NGS) has shown great promise in the analysis of DNA from degraded, limited, and mixed samples often encountered in forensic cases [1,2,3]. NGS platforms produce millions of reads for sequencing multiple samples in a single sequencing run, thereby making it possible for sequencing of the whole mitochondrial (mt) genome and proving valuable for analyzing nuclear single nucleotide polymorphism (SNPs) and short tandem repeats (STRs) [4,5,6,7]. NGS technologies are equipped with sequencing chemistry capable of generating 25 million–3 billion reads per sequencing run with an output of ~15–600 Gb file size, providing sufficient sequencing. NGS analysis of STR polymorphism has revealed additional sequence polymorphisms within tandem repeats not identified by conventional capillary electrophoresis approaches [9,10].
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