Construction of a library of cloned short tandem repeat (STR) alleles as universal templates for allelic ladder preparation

Wang, Le; Zhao, Xing‐Ming; Ye, Jian; Liu, Jinjie; Chen, Ting; Bai, Xue; Zhang, Jian; Ou, Yuan; Hu, Lan; Jiang, Bowei; Wang, Feng

doi:10.1016/j.fsigen.2014.06.005

Cited by 20 publications

(14 citation statements)

References 22 publications

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“…To examine whether this discordance occurred at the sequencing stage or the data analysis process, we reanalyzed the FASTQ file of the sample with Next GENe ® software. D7S820 allele sequences from our previous work were used as reference sequences. Alignment results indicated an adenine insertion before the consecutive 9 GATA repeats (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These data were used for calculating statistical parameters including depth of coverage (DoC), allele coverage ratio (ACR), and sequence coverage ratio (SCR) as described . FASTQ files of several samples were analyzed using Next GENe v.2.4.1.2 software (SoftGenetics, State College, PA) with allele sequences from our previous publication as the reference sequences . Matching requirement was set to be that no <12 sequential bases and 85% of the whole sequence to find its location.…”

Section: Methodsmentioning

confidence: 99%

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Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems,

Wang

Chen

et al. 2018

Journal of Forensic Sciences

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Next-generation sequencing (NGS) has been used to genotype forensic short tandem repeat (STR) markers for individual identification and kinship analysis. STR data from several NGS platforms have been published, but forensic application trials using the Ion S5™ XL system have not been reported. In this work, we report sensitivity, reproducibility, mixture, simulated degradation, and casework sample data on the Ion Chef™ and S5™ XL systems using an early access 25-plex panel. Sensitivity experiments showed that over 97% of the alleles were detectable with down to 62 pg input of genomic DNA. In mixture studies, alleles from minor contributors were correctly assigned at 1:9 and 9:1 ratios. NGS successfully gave 12 full genotype results from 13 challenging casework samples, compared with five full results using the CE platform. In conclusion, the Ion Chef™ and the Ion S5™ XL systems provided an alternative and promising approach for forensic STR genotyping.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems,

Wang

Chen

et al. 2018

Journal of Forensic Sciences

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“…The PAGE purification method has been the most widespread approach used in forensics to date, however it may be seen as low throughput, rate limiting for DNA recovery and not ideal for long‐term storage of the individual allele isolates. Some methods have therefore omitted the PAGE purification step [8,9] and the locus specific ladder is produced directly after the first PCR amplification from genomic DNA. Subsequently, the pooled alleles’ PCR mixture is diluted and re‐amplified for enrichment [10,11].…”

Section: Introductionmentioning

confidence: 99%

“…Although this approach is less labor intensive and provides faster turnover, it demands frequent utilization of genomic DNA especially for the rare alleles that may become depleted. The cloning of each allele presents a promising alternative as the massive allele enrichment is suitable for large productions and long‐term storage [9,12,13]. The major advantage of the cloning method is that recombinant bacterial colonies can be stored in glycerol for long term [14] and plated for outgrowth when required.…”

Section: Introductionmentioning

confidence: 99%

“…In this report, we make technical recommendations for producing a balanced allelic ladder using the allele cloning approach. Although this approach has been previously recommended [9,12,13], the current study identified various technical challenges that can impact the balance of the ladder, the quality, and the production throughput. We propose a simplified workflow with quality control steps to address these challenges and yield a multilocus ladder applicable for forensic applications.…”

Section: Introductionmentioning

confidence: 99%

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Allelic ladder production for a 10 locus Y‐chromosome DNA profiling system

Kasu

Fraser²,

Lesaoana

et al. 2020

Separation Science Plus

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The allelic ladder component of genotyping systems is of utmost importance for accurate allele designation and sizing precision quality assurances by capillary electrophoresis. The production process for ladders also demands adherence to specific technical criteria in order to pass as a reliable profiling system. The conventional methodology for ladder production includes allele isolation by polyacrylamide gel electrophoresis purification followed by amplification of each allele isolate using the polymerase chain reaction. Alternatively, the allele cloning approach has long been promoted as being less labor intensive, higher throughput, and more suitable for long‐term storage of the allele isolates. In this study, we present a workflow to produce a short tandem repeat allelic ladder using a cloning approach. We identified the key factors that may impact the quality of the ladder and provide recommendations to resolve these challenges. In this study, 143 alleles from 10 Y‐chromosome short tandem repeat loci were cloned and amplified individually from plasmid extracts. Amplicons were assessed using quality assurance criteria for selection of polymerase chain reaction products and dilution factors to produce, first, a balanced ladder for each locus, and then a final ladder compiled from each individual locus ladder. The final product was suitable for forensic applications.

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Development of a new 25plex STRs typing system for forensic application

et al. 2019

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We have developed a novel STR 25‐plex florescence multiplex‐STR kit (DNATyper25) to genotype 23 autosomal and two sex‐linked loci for forensic applications and paternity analysis. Of the 23 autosomal loci, 20 are non‐CODIS. The sex‐linked markers include a Y‐STR locus (DYS391) and the Amelogenin gene. We present developmental validation studies to show that the DNATyper25 kit is reproducible, accurate, sensitive, and robust. Sensitivity testing showed that full profiles were achieved with as low as 125 pg of human DNA. Specificity testing demonstrated a lack of cross reactivity with a variety of commonly encountered non‐human DNA contaminants. Stability testing showed that full profiles were obtained with humic acid concentration ≤60 ng/μL and hematin concentration <400 μM. For forensic evaluation, the 23 autosomal STRs followed the Hardy–Weinberg equilibrium. In an analysis of 509 Chinese (CN) Hans, we detected a combined total of 181 alleles at the 23 autosomal STR loci. Since these autosomal STRs are independent from one another, PM was 8.4528 × 10−22, TDP was 0.999 999 999 999 999 999 999, CEP was 0.999 999 8395. The forensic efficiency parameters demonstrated that these autosomal STRs are highly polymorphic and informative in the Han population of China. We performed population comparisons and showed that the Northern CN Han has a close genetic relationship with the Luzhou Han, Tujia, and Bai populations. We propose that the DNATyper25 kit will be useful for cases where paternity analysis is difficult and for situations where DNA samples are limited in quantity and low in quality.

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Construction of a library of cloned short tandem repeat (STR) alleles as universal templates for allelic ladder preparation

Cited by 20 publications

References 22 publications

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems,

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems,

Allelic ladder production for a 10 locus Y‐chromosome DNA profiling system

Development of a new 25plex STRs typing system for forensic application

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