Molecular diagnosis utility of multiplex ligation-dependent probe amplification

Chou, Lan-Szu; Lyon, Elaine; Mao, Rong

doi:10.1517/17530059.2.4.373

Cited by 5 publications

(1 citation statement)

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“…One of the limitations of both Sanger sequencing and MLPA analysis relates to allele dropout, which pertains to the inability of a PCR primer, sequencing primer, or an MLPA probe to bind and subsequently amplify one of the two alleles that may contain a sequence variant or a polymorphism, 15,16 thereby resulting in the reduced sensitivity. Allele dropout is effectively abolished in this NGS pipeline due to the redundancy that is introduced by the Roche/ Nimblegen library preparation protocol, including random genomic fragmentation and densely overlapped DNA library capture probes (up to two million unique probes per design).…”

Section: Clinical Ngs Pipeline In Targeted Gene Panel Analysismentioning

confidence: 99%

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

Schenkel

Kerkhof

Stuart

et al. 2016

The Journal of Molecular Diagnostics

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Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1/BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5' and 3' of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective (n = 95) and prospective (n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.

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Section: Clinical Ngs Pipeline In Targeted Gene Panel Analysismentioning

confidence: 99%