Dataset Information

A novel long-range PCR sequencing method for genetic analysis of the entire PKD1 gene.

ABSTRACT: Genetic testing of PKD1 and PKD2 is useful for the diagnosis and prognosis of autosomal dominant polycystic kidney disease; however, analysis is complicated by the large transcript size, the complexity of the gene region, and the high level of gene variations. We developed a novel mutation screening assay for PKD1 by directly sequencing long-range (LR) PCR products. By using this method, the entire PKD1 coding region was amplified by nine reactions, generating product sizes from 2 to 6 kb, circumventing the need for specific PCR amplification of individual exons. This method was compared with direct sequencing used by a reference laboratory and the SURVEYOR-WAVE Nucleic Acid High Sensitivity Fragment Analysis System (Transgenomic) screening method for five patients with autosomal dominant polycystic kidney disease. A total of 53 heterozygous genetic changes were identified by LR PCR sequencing, including 41 (of 42) variations detected by SURVEYOR nuclease and all 32 variations reported by the reference laboratory, detecting an additional 12 intronic changes not identified by the other two methods. Compared with the reference laboratory, LR PCR sequencing had a sensitivity of 100%, a specificity of 98.5%, and an accuracy of 98.8%; compared with the SURVEYOR-WAVE method, it had a sensitivity of 97.1%, a specificity of 100%, and an accuracy of 99.4%. In conclusion, LR PCR sequencing was superior to the direct sequencing and screening methods for detecting genetic variations, achieving high sensitivity and improved intronic coverage with a faster turnaround time and lower costs, and providing a reliable tool for complex genetic analyses.

SUBMITTER: Tan YC

PROVIDER: S-EPMC3391417 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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A novel long-range PCR sequencing method for genetic analysis of the entire PKD1 gene.

Tan Ying-Cai YC Michaeel Alber A Blumenfeld Jon J Donahue Stephanie S Parker Tom T Levine Daniel D Rennert Hanna H

The Journal of molecular diagnostics : JMD 20120516 4

Genetic testing of PKD1 and PKD2 is useful for the diagnosis and prognosis of autosomal dominant polycystic kidney disease; however, analysis is complicated by the large transcript size, the complexity of the gene region, and the high level of gene variations. We developed a novel mutation screening assay for PKD1 by directly sequencing long-range (LR) PCR products. By using this method, the entire PKD1 coding region was amplified by nine reactions, generating product sizes from 2 to 6 kb, circu ...[more]

PMID: 22608885

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Project description:BackgroundShort read sequencing has been used extensively to decipher the genome diversity of human cytomegalovirus (HCMV) strains, but falls short to reveal individual genomes in mixed HCMV strain populations. Novel third-generation sequencing platforms offer an extended read length and promise to resolve how distant polymorphic sites along individual genomes are linked. In the present study, we established a long amplicon PacBio sequencing workflow to identify the absolute and relative quantities of unique HCMV haplotypes spanning over multiple hypervariable sites in mixtures. Initial validation of this approach was performed with defined HCMV DNA templates derived from cell-culture enriched viruses and was further tested for its suitability on patient samples carrying mixed HCMV infections.ResultsTotal substitution and indel error rate of mapped reads ranged from 0.17 to 0.43% depending on the stringency of quality trimming. Artificial HCMV DNA mixtures were correctly determined down to 1% abundance of the minor DNA source when the total HCMV DNA input was 4 × 104 copies/ml. PCR products of up to 7.7 kb and a GC content < 55% were efficiently generated when DNA was directly isolated from patient samples. In a single sample, up to three distinct haplotypes were identified showing varying relative frequencies. Alignments of distinct haplotype sequences within patient samples showed uneven distribution of sequence diversity, interspersed by long identical stretches. Moreover, diversity estimation at single polymorphic regions as assessed by short amplicon sequencing may markedly underestimate the overall diversity of mixed haplotype populations.ConclusionsQuantitative haplotype determination by long amplicon sequencing provides a novel approach for HCMV strain characterisation in mixed infected samples which can be scaled up to cover the majority of the genome by multi-amplicon panels. This will substantially improve our understanding of intra-host HCMV strain diversity and its dynamic behaviour.

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

A novel long-range PCR sequencing method for genetic analysis of the entire PKD1 gene.

Publications

A novel long-range PCR sequencing method for genetic analysis of the entire PKD1 gene.

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