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Simple and efficient germline copy number variant visualization method for the Ion AmpliSeq™ custom panel.

ABSTRACT: BACKGROUND:Recent advances in molecular genetic analysis using next-generation sequencing (NGS) have drastically accelerated the identification of disease-causing gene mutations. Most next-generation sequencing analyses of inherited diseases have mainly focused on single-nucleotide variants and short indels, although, recently, structure variations including copy number variations have come to be considered an important cause of many different diseases. However, only a limited number of tools are available for multiplex PCR-based target genome enrichment. METHODS:In this paper, we reported a simple and efficient copy number variation visualization method for Ion AmpliSeq™ target resequencing data. Unlike the hybridization capture-based target genome enrichment system, Ion AmpliSeq™ reads are multiplex PCR products, and each read generated by the same amplicon is quite uniform in length and position. Based on this feature, the depth of coverage information for each amplicon included in the barcode/amplicon coverage matrix file was used for copy number detection analysis. We also performed copy number analysis to investigate the utility of this method through the use of positive controls and a large Japanese hearing loss cohort. RESULTS:Using this method, we successfully confirmed previously reported copy number loss cases involving the STRC gene and copy number gain in trisomy 21 cases. We also performed copy number analysis of a large Japanese hearing loss cohort (2,475 patients) and identified many gene copy number variants. The most prevalent copy number variation was STRC gene copy number loss, with 129 patients carrying this copy number variation. CONCLUSION:Our copy number visualization method for Ion AmpliSeq™ data can be utilized in efficient copy number analysis for the comparison of a large number of samples. This method is simple and requires only easy calculations using standard spread sheet software.

SUBMITTER: Nishio SY

PROVIDER: S-EPMC6081219 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Simple and efficient germline copy number variant visualization method for the Ion AmpliSeq™ custom panel.

Nishio Shin-Ya SY Moteki Hideaki H Usami Shin-Ichi SI

Molecular genetics & genomic medicine 20180406

<h4>Background</h4>Recent advances in molecular genetic analysis using next-generation sequencing (NGS) have drastically accelerated the identification of disease-causing gene mutations. Most next-generation sequencing analyses of inherited diseases have mainly focused on single-nucleotide variants and short indels, although, recently, structure variations including copy number variations have come to be considered an important cause of many different diseases. However, only a limited number of ...[more]

PMID: 29633566

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Project description:Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.

Project description:Ataxia-telangiectasia (A-T), or Louis-Bar syndrome, is a rare neurodegenerative disorder associated with immunodeficiency. For families with at least one affected child, timely A-T genotyping during any subsequent pregnancy allows the parents to make an informed decision about whether to continue to term when the fetus is affected. Mutations in the ATM gene, which is 150?kb long, give rise to A-T; more than 600 pathogenic variants in ATM have been characterized since 1990 and new mutations continue to be discovered annually. Therefore, limiting genetic screening to previously known SNPs by PCR or hybridization with microarrays may not identify the specific pathogenic genotype in ATM for a given A-T family. However, recent developments in next-generation sequencing technology offer prompt high-throughput full-length sequencing of genomic fragments of interest. This allows the identification of the whole spectrum of mutations in a gene, including any novel ones. We report two A-T families with affected children and current pregnancies. Both families are consanguineous and originate from Caucasian regions of Russia and Azerbaijan. Before our study, no ATM mutations had been identified in the older children of these families. We used ion semiconductor sequencing and an Ion AmpliSeq™ Inherited Disease Panel to perform complete ATM gene sequencing in a single member of each family. Then we compared the experimentally determined genotype with the affected/normal phenotype distribution in the whole family to provide unambiguous evidence of pathogenic mutations responsible for A-T. A single novel SNP was allocated to each family. In the first case, we found a mononucleotide deletion, and in the second, a mononucleotide insertion. Both mutations lead to truncation of the ATM protein product. Identification of the pathogenic mutation in each family was performed in a timely fashion, allowing the fetuses to be tested and diagnosed. The parents chose to continue with both pregnancies as both fetuses had a healthy genotype and thus were not at risk of A-T.

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Dataset Information

Simple and efficient germline copy number variant visualization method for the Ion AmpliSeq™ custom panel.

Publications

Simple and efficient germline copy number variant visualization method for the Ion AmpliSeq™ custom panel.

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