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Investigation of the STR loci noise distributions of PowerSeq™ Auto System.

ABSTRACT: AIM:To characterize the noise and stutter distribution of 23 short tandem repeats (STRs) included in the PowerSeqTM Auto System. METHODS:Raw FASTQ files were analyzed using STRait Razor v2s to display alleles and coverage. The sequence noise was divided into several categories: noise at allele position, noise at -1 repeat position, and artifact. The average relative percentages of locus coverage for each noise, stutter, and allele were calculated from the samples used for this locus noise analysis. RESULTS:Stutter products could be routinely observed at the -2 repeat position, -1 repeat position, and +1 repeat position of alleles. Sequence noise at the allele position ranged from 10.22% to 28.81% of the total locus coverage. At the allele position, individual noise reads were relatively low. CONCLUSION:The data indicate that noise generally will be low. In addition, the PowerSeqTM Auto System could capture nine flanking region single nucleotide polymorphisms (SNPs) that would not be observed by other current kits for massively parallel sequencing (MPS) of STRs.

SUBMITTER: Zeng X

PROVIDER: S-EPMC5470120 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Investigation of the STR loci noise distributions of PowerSeq™ Auto System.

Zeng Xiangpei X King Jonathan L JL Budowle Bruce B

Croatian medical journal 20170601 3

<h4>Aim</h4>To characterize the noise and stutter distribution of 23 short tandem repeats (STRs) included in the PowerSeqTM Auto System.<h4>Methods</h4>Raw FASTQ files were analyzed using STRait Razor v2s to display alleles and coverage. The sequence noise was divided into several categories: noise at allele position, noise at -1 repeat position, and artifact. The average relative percentages of locus coverage for each noise, stutter, and allele were calculated from the samples used for this loc ...[more]

PMID: 28613038

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Project description:Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina's MiSeq FGx MPS system and ForenSeq™ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina's FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.

Dataset Information

Investigation of the STR loci noise distributions of PowerSeq™ Auto System.

Publications

Investigation of the STR loci noise distributions of PowerSeq™ Auto System.

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