Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa

Abstract

Massively parallel sequencing (MPS) is an emerging technology in the field of forensic genetics that provides distinct advantages compared to capillary electrophoresis. This study offers a proof of concept that MPS technologies can be applied to genotype autosomal STRs in Cannabis sativa. A custom panel for MPS was designed to interrogate 12 cannabis-specific STR loci by sequence rather than size. A simple workflow was implemented to integrate the custom PCR multiplex into a workflow compatible with the Ion Plus Fragment Library Kit, Ion Chef, and Ion S5 System. For data sorting and sequence analysis, a custom configuration file was designed for STRait Razor v3 to parse and extract STR sequence data. This study represents a preliminary investigation of sequence variation for 12 autosomal STR loci in 16 cannabis samples. Full concordance was observed between the MPS and CE data. Results revealed intra-repeat variation in eight loci where the nominal or size-based allele was identical, but variances were discovered in the sequence of the flanking region. Although only a small number of cannabis samples were evaluated, this study demonstrates that more informative STR data can be obtained via MPS.

CSYseq: The first Y-chromosome sequencing tool typing a large number of Y-SNPs and Y-STRs to unravel worldwide human population genetics

“…Currently, the two most comprehensive commercial CE-kits for Y-STR DNA profiling are the PowerPlex Y23 (23 Y-STRs, Promega) and the Yfiler Plus PCR Amplification Kit (27 Y-STRs, Applied Biosystems) [ 33 ]. With MPS, a large number of markers (both Y-SNPs and Y-STRs) can be analyzed simultaneously, reaching a higher discrimination capacity and wider range of applications [ 32 , 34 ]. To date, several MPS panels are already commercialized for SNP identity and ancestry analysis as well as STR marker DNA profiling.…”

Massively parallel sequencing of Cannabis sativa chloroplast hotspots for forensic typing

“…Houston et al reported an MPS panel for the Ion S5™ consisting of twelve autosomal STRs in Cannabis sativa (marijuana). Results showed concordance with CE methods, and isoalleles were found at eight of the loci, providing a higher discriminatory power compared to CE (Houston et al, 2018a , b ). MPS has been also been used for DNA barcoding studies in animals and has shown better recovery, reduced costs, and faster processing times compared to traditional Sanger sequencing (Shokralla et al 2015 ).…”

“…Chilean samples were provided by collaborators in the form of DNA extracts. Chloroplast DNA was quantified using a real-time PCR method reported by Houston et al (Houston et al, 2018a , b ).…”

“…In forensics, MPS assays have been used for human identification purposes, including sequencing autosomal STRs and single nucleotide polymorphisms (SNPs) (Eduardoff et al 2015 ; Guo et al 2017 ; Kim et al 2016 ; Seo et al 2013 ; Wang et al 2017 ), mitochondrial DNA analysis (Davis et al 2015 ), phenotype prediction (Mehta et al 2016 ), and other purposes (Bruijns et al 2018 ; Budowle et al 2017 ). While much of the forensic research on MPS has focused on human DNA, its use for forensic plant science has recently been investigated (Houston et al, 2018a , b ). Houston et al reported an MPS panel for the Ion S5™ consisting of twelve autosomal STRs in Cannabis sativa (marijuana).…”

The forensic landscape and the population genetic analyses of Hainan Li based on massively parallel sequencing DNA profiling

“…With the observations of the sequence variations which derived high polymorphisms of some autosomal STR loci in Hainan Li population and other populations that the variations also existed [84,[111][112][113][114][115][116][117][118][119][120][121][122][123][124], it indicated that the sequence variation-derived high polymorphisms widely existed in different populations, and the genetic diversity of the same locus in different populations were diverse under some circumstances. It could be explained by the relatively isolations between populations from the cultural, geographical and social differences, and genetic mutations accumulated over many years in populations.…”