Dataset Information

Streamlining Genetic Diagnosis With Long-Range Polymerase Chain Reaction (PCR)-Based Next-Generation Sequencing for Type I and Type II Collagenopathies.

ABSTRACT: In the practice of clinical genetics, gene testing is usually guided by clinical diagnosis. When dealing with rare diseases, it is often necessary to create new test systems. The handling of a gene with a substantial number of exons poses a challenge both in sequential Sanger sequencing for each exon, and in the setup of capture probes to each exon for next-generation sequencing (NGS). We present very long amplicon sequencing (vLAS), an optimized long-range polymerase chain reaction (PCR)-based NGS method that overcomes this challenge. By utilizing approximately 20 Kb long PCR products and short-read NGS, vLAS is emerging as a highly adaptable and effective solution, especially for genes with numerous exons concentrated in a limited genomic region. Here, we demonstrate vLAS in the analysis of five patients with type I and two with type II collagenopathies. The integration of user-friendly NGS methods into genetic diagnosis enhances the practicality of clinical genetics.

SUBMITTER: Niida Y

PROVIDER: S-EPMC10788244 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Streamlining Genetic Diagnosis With Long-Range Polymerase Chain Reaction (PCR)-Based Next-Generation Sequencing for Type I and Type II Collagenopathies.

Niida Yo Y Togi Sumihito S Ura Hiroki H

Cureus 20231213 12

In the practice of clinical genetics, gene testing is usually guided by clinical diagnosis. When dealing with rare diseases, it is often necessary to create new test systems. The handling of a gene with a substantial number of exons poses a challenge both in sequential Sanger sequencing for each exon, and in the setup of capture probes to each exon for next-generation sequencing (NGS). We present very long amplicon sequencing (vLAS), an optimized long-range polymerase chain reaction (PCR)-based ...[more]

PMID: 38226081

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Project description:BackgroundDuring the last decade, remarkable progress with massive sequencing has been made in the identification of disease-associated genes for AIDs using next-generation sequencing technologies (NGS). An international group of experts described the ideal genetic screening method which should give information about SNVs, InDels, Copy Number Variations (CNVs), GC rich regions. We aimed to develop and validate a molecular diagnostic method in conjunction with the NGS platform as an inexpensive, extended and uniform coverage and fast screening tool which consists of nine genes known to be associated with various AIDs.MethodsFor the validation of basic and expanded panels, long-range multiplex models were setup on healthy samples without any known variations for MEFV, MVK, TNFRSF1A, NLRP3, PSTPIP1, IL1RN, NOD2, NLRP12 and LPIN2 genes. Patients with AIDs who had already known causative variants in these genes were sequenced for analytical validation. As a last step, multiplex models were validated on patients with pre-diagnosis of AIDs. All sequencing steps were performed on the Illumina NGS platform. Validity steps included the selection of related candidate genes, primer design, development of screening methods, validation and verification of the product. The GDPE (Gentera) bioinformatics pipeline was followed.ResultsAlthough there was no nonsynonymous variation in 21 healthy samples, 107 synonymous variant alleles and some intronic and UTR variants were detected. In 10 patients who underwent analytical validation, besides the 11 known nonsynonymous variant alleles, 11 additional nonsynonymous variant alleles and a total of 81 synonymous variants were found. In the clinical validation phase, 46 patients sequenced with multiplex panels, genetic and clinical findings were combined for diagnosis.ConclusionIn this study, we describe the development and validation of an NGS-based multiplex array enabling the "long-amplicon" approach for targeted sequencing of nine genes associated with common AIDs. This screening tool is less expensive and more comprehensive compared to other methods and more informative than traditional sequencing. The proposed panel offers advantages to WES or hybridization probe equivalents in terms of CNV analysis, high sensitivity and uniformity, GC-rich region sequencing, InDel detection and intron covering.

Dataset Information

Streamlining Genetic Diagnosis With Long-Range Polymerase Chain Reaction (PCR)-Based Next-Generation Sequencing for Type I and Type II Collagenopathies.

Publications

Streamlining Genetic Diagnosis With Long-Range Polymerase Chain Reaction (PCR)-Based Next-Generation Sequencing for Type I and Type II Collagenopathies.

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