High Throughput Sequencing of Forensically Relevant Single Nucleotide Polymorphisms
Open Access
- Author:
- Shugarts, Nathan Maxwell
- Area of Honors:
- Forensic Science
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Jenifer A L Smith, Thesis Supervisor
Jenifer A L Smith, Thesis Honors Advisor
Dr. Mitchell Mark Holland, Faculty Reader - Keywords:
- forensic
molecular
biology
single
nucleotide
polymorphism
history
high
throughput
sequencing
MiSeq
Illumina
ForenSeq - Abstract:
- In order to identify the source of a body fluid stain, human DNA can be characterized, or “typed”, by analyzing alleles of short tandem repeat (STR) segments that vary in number at and between specific chromosomal loci. Multiplex PCR creates fragments that are separated and detected via capillary electrophoresis (CE), based on length alone. The result is an STR profile that lists the alleles for each locus. This profile can be compared to either profiles obtained from reference samples or those stored in a DNA database. Often the comparison does not yield a match and the source of the stain remains unidentified. Regions of DNA that contain areas of single nucleotide polymorphism (SNP) have been identified that may provide “lead” information about the lineage, ancestry, and/or phenotype of the source. However, the genotyping of SNPs has historically been a laborious process, especially when dealing with profile mixture interpretation, discouraging their use in forensic science laboratories. With next generation sequencing (NGS) of DNA, characterized by its massively parallel sequencing technologies, the process of SNP sequencing has been simplified. In this study the MiSeq FGx (Illumina) NGS platform, employing sequencing by synthesis (SBS) technology, was used to interrogate SNP regions of interest targeted by the ForenSeq DNA Signature Prep Kit (Illumina). Validation experiments were executed that tested kit reproducibility and sensitivity, as well as concordance with other methods. The results of these validation studies may help the forensic science community determine whether or not the MiSeq FGx and ForenSeq DNA Signature Prep Kit are robust and reliable enough for use on casework samples. Additional validation studies involving mixture interpretation and mock case samples must still be executed.