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

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Abstract

Aim

To characterize the noise and stutter distribution of 23 short tandem repeats (STRs) included in the PowerSeq ^TM 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 PowerSeq ^TM 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.

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Most cited references 21

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Slippage synthesis of simple sequence DNA.

D Tautz, C Schlötterer (1992)

The analysis of slippage synthesis of simple sequence DNA in vitro sheds some light on the question of how simple sequences arise in vivo. We show that it is possible to synthesize all types of repetitious di- and trinucleotide motifs starting from short primers and a polymerase in vitro. The rate of this synthesis depends on a sequence specific slippage rate, but is independent of the length of the fragments being synthesized. This indicates that only the ends of the DNA fragments are involved in determining this rate and that slippage is accordingly a short range effect. Slippage synthesis occurs also on a fixed template where only one strand is free to move, a situation which resembles chromosome replication in vivo. It seems therefore likely that slippage during replication is the cause of the observed length polymorphism of simple sequence stretches between individuals of a population.

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Replication slippage involves DNA polymerase pausing and dissociation.

D Canceill, E Viguera, S D Ehrlich (2001)

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.

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Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories.

Anne C Jäger, Michelle L Alvarez, Carey P Davis … (2017)

Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities.

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Author and article information

Journal

Journal ID (nlm-ta): Croat Med J

Journal ID (iso-abbrev): Croat. Med. J

Journal ID (publisher-id): CMJ

Title: Croatian Medical Journal

Publisher: Croatian Medical Schools

ISSN (Print): 0353-9504

ISSN (Electronic): 1332-8166

Publication date (Print): June 2017

Volume: 58

Issue: 3

Pages: 214-221

Affiliations

[1 ]Center for Human Identification, University of North Texas Health Science Center, Fort Worth, TX, USA

[2 ]Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia

Author notes

Correspondence to: Xiangpei Zeng Center for Human Identification University of North Texas Health Science Center 3500 Camp Bowie Boulevard Fort Worth, TX 76107, USA  Xiangpei.Zeng@ 123456unthsc.edu

Article

Publisher ID: CroatMedJ_58_0214

DOI: 10.3325/cmj.2017.58.214

PMC ID: 5470120

PubMed ID: 28613038

SO-VID: e54a93fd-c9c3-4fc7-b948-111df010becc

License:

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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

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Abstract

Aim

Methods

Results

Conclusion

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Universal stem cells

Most cited references 21

Slippage synthesis of simple sequence DNA.

Replication slippage involves DNA polymerase pausing and dissociation.

Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories.

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