Allele typing of short tandem repeats by capillary electrophoresis

Tagliabracci, Adriano; Buscemi, Loredana; Sassaroli, Corrado; Paoli, Massimo De; Rodriguez, Daniele

doi:10.1007/s004140050274

Cited by 12 publications

(4 citation statements)

References 40 publications

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“…The STRs are usually amplified using commercial multiplex PCR kits and the alleles are detected using capillary electrophoresis (CE) by comparing the amplified products with an allelic ladder. Under the recommended CE conditions, the variation in sizing precision is approximately 0.15-0.25 nt [2][3][4][5], which is sufficient to allow phenotyping of alleles that differ in length by one nucleotide. However, others have reported variations up to 0.8 for the longest alleles of the FGA locus [6].…”

Section: Introductionmentioning

confidence: 99%

Non-uniform phenotyping of D12S391 resolved by second generation sequencing

Dalsgaard

Rockenbauer

Buchard

et al. 2014

Forensic Science International: Genetics

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Non-uniform phenotyping of five case work samples were observed in the D12S391 locus. The samples were typed at least twice with the AmpFℓSTR NGM SElect PCR Amplification Kit and different alleles were called with GeneMapper ID-X in the different experiments. Detailed analyses of the electropherograms suggested that the individuals were heterozygous with two alleles that differed in size by one nucleotide. This was confirmed by amplifying the samples with the PowerPlex ESX 17 system. D12S391 is a complex STR with variable numbers of AGAT and AGAC repeats. Second generation sequencing revealed that separation of two alleles differing by one nucleotide in length was poor if the number of AGAT repeats in the short allele was higher than in the long allele. A total of 45 individuals with microvariants or off-ladder alleles in D12S391 were sequenced. Thirty different alleles were detected and sixteen of these were not previously reported.

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Section: Introductionmentioning

confidence: 99%

Non-uniform phenotyping of D12S391 resolved by second generation sequencing

Dalsgaard

Rockenbauer

Buchard

et al. 2014

Forensic Science International: Genetics

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“…Traditionally, TR analysis has been carried out via restriction fragment length polymorphism (RFLP) analysis [8] or PCR amplification of the target loci followed by fragment length analysis [9]. These techniques are only applicable to a specific target region and not scalable to high-throughput analysis, which limits the possibility of genome-wide TR analysis.…”

Section: Introductionmentioning

confidence: 99%

High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing

Ganesamoorthy

Yan

Murigneux

et al. 2019

Preprint

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3Tandem repeats (TRs) are highly prone to variation in copy numbers due to their repetitive and 2 4 unstable nature, which makes them a major source of genomic variation between individuals. 5However, population variation of TRs have not been widely explored due to the limitations of 2 6 existing tools, which are either low-throughput or restricted to a small subset of TRs. Here, we 2 7used SureSelect targeted sequencing approach combined with Nanopore sequencing to 2 8 overcome these limitations. We achieved an average of 3062-fold target enrichment on a panel 2 9 of 142 TR loci, generating an average of 97X sequence coverage on 7 samples utilizing 2 3 0 MinION flow-cells with 200ng of input DNA per sample. We identified a subset of 110 TR loci 3 1with length less than 2kb, and GC content greater than 25% for which we achieved an average 3 2 genotyping rate of 75% and increasing to 91% for the highest-coverage sample. Alleles 3 3 estimated from targeted long-read sequencing were concordant with gold standard PCR sizing 3 4 analysis and moreover highly correlated with alleles estimated from whole genome long-read 3 5sequencing. We demonstrate a targeted long-read sequencing approach that enables 3 6

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“…Traditionally, TR analysis has been carried out via restriction fragment length polymorphism (RFLP) analysis in which restriction enzymes are designed to fragment a target region, and genotyping was carried out by separation of fragments on a gel [ 12 ]. Recently, PCR amplification of the target loci, followed by capillary electrophoresis analysis was used to determine the fragment length of the alleles [ 13 ]. However, these techniques are only applicable to a specific target region and not scalable to high-throughput analysis.…”

Section: Introductionmentioning

confidence: 99%

GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing

et al. 2018

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BackgroundTandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations. We report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely – GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation.ResultsWe used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68 and 83% for capture sequence data and 200X WGS data respectively, improving to 87 and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25, 14, 12 and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results.ConclusionsThe novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.Electronic supplementary materialThe online version of this article (10.1186/s12859-018-2282-3) contains supplementary material, which is available to authorized users.

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Allele typing of short tandem repeats by capillary electrophoresis

Cited by 12 publications

References 40 publications

Non-uniform phenotyping of D12S391 resolved by second generation sequencing

Non-uniform phenotyping of D12S391 resolved by second generation sequencing

High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing

GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing

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