Massively parallel sequencing of customised forensically informative SNP panels on the MiSeq.
Forensic DNA-based intelligence, or forensic DNA phenotyping, utilises SNPs to infer the biogeographical ancestry and externally visible characteristics of the donor of evidential material. SNaPshot®is a commonly employed forensic SNP genotyping technique, which is limited to multiplexes of 30-40 SNPs in a single reaction and prone to PCR contamination. Massively parallel sequencing has the ability to genotype hundreds of SNPs in multiple samples simultaneously by employing an oligonucleotide sample barcoding strategy. This study of the Illumina MiSeq massively parallel sequencing platform analysed 136 unique SNPs in 48 samples from SNaPshot PCR amplicons generated by five established forensic DNA phenotyping assays comprising the SNPforID 52-plex, SNPforID 34-plex, Eurasiaplex, Pacifiplex and IrisPlex. Approximately 3 GB of sequence data were generated from two MiSeq flow cells and profiles were obtained from just 0.25 ng of DNA. Compared with SNaPshot, an average 98% genotyping concordance was achieved. Our customised approach was successful in attaining SNP profiles from extremely degraded, inhibited, and compromised casework samples. Heterozygote imbalance and sequence coverage in negative controls highlight the need to establish baseline sequence coverage thresholds and refine allele frequency thresholds. This study demonstrates the potential of the MiSeq for forensic SNP analysis.
National Centre for Forensic Studies, Faculty of ESTeM, University of Canberra, Canberra, Australia. bhavik.mehta@hotmail.com.
Bhavik
B
Mehta
Office of the Chief Forensic Scientist, Victoria Police Forensic Services Department, Melbourne, Australia.
Runa
R
Daniel
Forensic Genetics Unit, Institute of Legal Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain.
Chris
C
Phillips
Department of Animal, Plant and Soil Sciences, La Trobe University, Melbourne, Australia.
Stephen
S
Doyle
Illumina Inc, San Diego, CA, USA.
Gareth
G
Elvidge
National Centre for Forensic Studies, Faculty of ESTeM, University of Canberra, Canberra, Australia.
Dennis
D
McNevin
D004247:DNA / Q000032:analysis* / Q000235:genetics; D005260:Female; D053803:Forensic Genetics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D006812:Humic Substances; D008297:Male; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
D006812:Humic Substances; D004247:DNA
Forensic DNA genotyping; Illumina MiSeq; Next generation sequencing (NGS); SNPs; SNaPshot