Developmental and internal validation of a novel 13 loci STR multiplex method for Cannabis sativa DNA profiling

Houston, Rachel; Birck, Matthew; Hughes‐Stamm, Sheree; Gangitano, David

doi:10.1016/j.legalmed.2017.03.001

Cited by 23 publications

(40 citation statements)

References 16 publications

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“…This information can greatly increase the power of discrimination through intra‐repeat variation and SNPs within the flanking region. The authors’ previously published CE based 13‐STR multiplex had a power of discrimination of 1 in 55 million . While the power of discrimination would drop to 1 in 39 million by eliminating the CS1 marker, the authors would expect this power of discrimination to increase with the extra discriminating power afforded by the sequence variants (isoalleles).…”

Section: Resultsmentioning

confidence: 99%

“…A custom AmpliSeq™ panel could not be designed as Cannabis sativa is not currently a supported species and a reference genome is not available. Instead, primer sequences (non‐fluorescent) from a previous CE method were used . Primer sequences and PCR parameters from the previously validated multiplex were used to ensure adequate amplification efficiency of all amplicons.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, primer concentrations were titrated according at Houston et al. to ensure a more balanced sequencing profile of the amplicons. To note, the hexanucleotide marker, CS1, was not included in MPS analysis due to the data analysis challenges posed by the marker's highly variable amplicon length and complexity of sequence.…”

Section: Methodsmentioning

confidence: 99%

“…Cannabis STR profiling was performed in a 13‐loci multiplex format using a previously validated method according to Houston et al. . All loci were identical to the MPS method with the addition of the highly complex marker, CS1.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, a previously validated STR multiplex for cannabis genetic identification was successfully incorporated into an MPS workflow . MPS performance including read depth, heterozygote balance, noise, and CE concordance was assessed.…”

Section: Introductionmentioning

confidence: 99%

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Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa

et al. 2018

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa

et al. 2018

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Development and validation of a forensic multiplex InDel assay: The AGCU InDel 60 kit

Liu

Jiang

et al. 2022

Electrophoresis

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Marker sets based on insertion/deletion polymorphisms (InDels) combine the characteristics of both short tandem repeats (STRs) and single nucleotide polymorphisms and have served as effective complementary or stand-alone systems for human identification in forensics. We developed a novel multiplex amplification detection system, designated the AGCU InDel 60 kit, containing 57 autosomal InDels, 2 Y-chromosomal InDels, and the amelogenin locus and validated the kit in a series of studies, which included tests of the PCR conditions; tests for sensitivity, species specificity, reproducibility, stability, and mock case samples; degradation studies; and a population study. The results indicated that the AGCU InDel 60 kit was accurate, specific, reproducible, stable, and robust. Complete DNA profiles were obtained even with 125 pg of human DNA. In tests of artificially degraded samples, we found that the number of alleles detected by the validated kit was considerably greater than that detected by the STRbased AGCU 21+1 kit, even as the degree of degradation increased. Additionally, 564 unrelated individuals from three Han groups were investigated using this novel system, and the values of combined power of discrimination and combined power of exclusion were not less than 1-4.9026 × 10 −24 and 1-3.1123 × 10 −5 , respectively. Thus, the results indicated that the novel kit was more powerful than the previous version of the InDel kit (the AGCU InDel 50 kit). Our results suggest that the AGCU InDel 60 kit can serve as an efficient tool for human forensics and a supplementary kit for population genetics research.

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Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database

et al. 2018

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As Cannabis sativa (marijuana) is a controlled substance in many parts of the world, the ability to track biogeographical origin of cannabis could provide law enforcement with investigative leads regarding its trade and distribution. Population substructure and inbreeding may cause cannabis plants to become more genetically related. This genetic relatedness can be helpful for intelligence purposes. Analysis of autosomal, chloroplast, and mitochondrial DNA allows for not only prediction of biogeographical origin of a plant but also discrimination between individual plants. A previously validated, 13-autosomal STR multiplex was used to genotype 510 samples. Samples were analyzed from four different sites: 21 seizures at the US-Mexico border, Northeastern Brazil, hemp seeds purchased in the US, and the Araucania area of Chile. In addition, a previously reported multi-loci system was modified and optimized to genotype five chloroplast and two mitochondrial markers. For this purpose, two methods were designed: a homopolymeric STR pentaplex and a SNP triplex with one chloroplast (Cscp001) marker shared by both methods for quality control. For successful mitochondrial and chloroplast typing, a novel real-time PCR quantitation method was developed and validated to accurately estimate the quantity of the chloroplast DNA (cpDNA) using a synthetic DNA standard. Moreover, a sequenced allelic ladder was also designed for accurate genotyping of the homopolymeric STR pentaplex. For autosomal typing, 356 unique profiles were generated from the 425 samples that yielded full STR profiles and 25 identical genotypes within seizures were observed. Phylogenetic analysis and case-to-case pairwise comparisons of 21 seizures at the US-Mexico border, using the Fixation Index (F ) as genetic distance, revealed the genetic association of nine seizures that formed a reference population. For mitochondrial and chloroplast typing, subsampling was performed, and 134 samples were genotyped. Complete haplotypes (STRs and SNPs) were observed for 127 samples. As expected, extensive haplotype sharing was observed; five distinguishable haplotypes were detected. In the reference population, the same haplotype was observed 39 times and two unique haplotypes were also detected. Haplotype sharing was observed between the US border seizures, Brazil, and Chile, while the hemp samples generated a distinct haplotype. Phylogenetic analysis of the four populations was performed, and results revealed that both autosomal and lineage markers could discern population substructure.

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Developmental and internal validation of a novel 13 loci STR multiplex method for Cannabis sativa DNA profiling

Cited by 23 publications

References 16 publications

Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa

Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa

Development and validation of a forensic multiplex InDel assay: The AGCU InDel 60 kit

Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database

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