Dataset Information

Accuracy and Performance Evaluation of Triplet Repeat Primed PCR as an Alternative to Conventional Diagnostic Methods for Fragile X Syndrome.

ABSTRACT:

Background

Conventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA and/or SB in terms of allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers.

Methods

From November 2013 to March 2018, 458 patients (326 males, 132 females) were simultaneously examined using FA and/or SB and TP-PCR by detecting CGG repeat numbers in FMR1 gene and diagnosed as per American College of Medical Genetics guidelines.

Results

The TP-PCR results showed high concordance with the FA and/or SB results for all three aspects (allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers). TP-PCR detected CGG expansions ≥200 in all full mutation (FM) allele cases in male patients, as well as both the normal allele (NL) and FM allele in female carriers. In premutation (PM) allele carriers, the TP-PCR results were consistent with the FA and/or SB results. In terms of zygosity concordance in female genetic carriers, 12 NL cases detected by TP-PCR showed a merged peak consisting of two close heterozygous peaks; however, this issue was resolved using a 10-fold dilution.

Conclusions

TP-PCR may serve as a reliable alternative method for FXS diagnosis.

SUBMITTER: Gu H

PROVIDER: S-EPMC7884195 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Publications

Accuracy and Performance Evaluation of Triplet Repeat Primed PCR as an Alternative to Conventional Diagnostic Methods for Fragile X Syndrome.

Gu Hyunjung H Kim Man Jin MJ Yang Dahae D Song Ji Yun JY Cho Sung Im SI Park Sung Sup SS Seong Moon-Woo MW

Annals of laboratory medicine 20210701 4

<h4>Background</h4>Conventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA ...[more]

PMID: 33536358

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Project description:The gut microbiome of animals is emerging as an important factor influencing ecological and evolutionary processes. A major bottleneck in obtaining microbiome data from large numbers of samples is the time-consuming laboratory procedures required, specifically the isolation of DNA and generation of amplicon libraries. Recently, direct PCR kits have been developed that circumvent conventional DNA extraction steps, thereby streamlining the laboratory process by reducing preparation time and costs. However, the reliability and efficacy of direct PCR for measuring host microbiomes have not yet been investigated other than in humans with 454 sequencing. Here, we conduct a comprehensive evaluation of the microbial communities obtained with direct PCR and the widely used Mo Bio PowerSoil DNA extraction kit in five distinct gut sample types (ileum, cecum, colon, feces, and cloaca) from 20 juvenile ostriches, using 16S rRNA Illumina MiSeq sequencing. We found that direct PCR was highly comparable over a range of measures to the DNA extraction method in cecal, colon, and fecal samples. However, the two methods significantly differed in samples with comparably low bacterial biomass: cloacal and especially ileal samples. We also sequenced 100 replicate sample pairs to evaluate repeatability during both extraction and PCR stages and found that both methods were highly consistent for cecal, colon, and fecal samples (rs > 0.7) but had low repeatability for cloacal (rs = 0.39) and ileal (rs = -0.24) samples. This study indicates that direct PCR provides a fast, cheap, and reliable alternative to conventional DNA extraction methods for retrieving 16S rRNA data, which can aid future gut microbiome studies. IMPORTANCE The microbial communities of animals can have large impacts on their hosts, and the number of studies using high-throughput sequencing to measure gut microbiomes is rapidly increasing. However, the library preparation procedure in microbiome research is both costly and time-consuming, especially for large numbers of samples. We investigated a cheaper and faster direct PCR method designed to bypass the DNA isolation steps during 16S rRNA library preparation and compared it with a standard DNA extraction method. We used both techniques on five different gut sample types collected from 20 juvenile ostriches and sequenced samples with Illumina MiSeq. The methods were highly comparable and highly repeatable in three sample types with high microbial biomass (cecum, colon, and feces), but larger differences and low repeatability were found in the microbiomes obtained from the ileum and cloaca. These results will help microbiome researchers assess library preparation procedures and plan their studies accordingly.

Dataset Information

Accuracy and Performance Evaluation of Triplet Repeat Primed PCR as an Alternative to Conventional Diagnostic Methods for Fragile X Syndrome.

Background

Methods

Results

Conclusions

Publications

Accuracy and Performance Evaluation of Triplet Repeat Primed PCR as an Alternative to Conventional Diagnostic Methods for Fragile X Syndrome.

Similar Datasets

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