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A comprehensive assessment of Next-Generation Sequencing variants validation using a secondary technology.

ABSTRACT: BACKGROUND:Recently, increasing innovations improved the accuracy of next generation sequencing (NGS) data. However, the validation of all NGS variants increased the cost and turn-around time of clinical diagnosis, and therefore limited the further development of clinical applications. We aimed to comprehensively assess the necessity of validating NGS variants. METHODS:Validation data of 7,601 NGS variants involving 1,045 genes were collected from 5,190 clinical samples and sequenced by one of five targeted capture panels and two NGS chemistries, respectively. These genes and variants were widely distributed in 24 human chromosomes and mitochondrial genome. Variants validation was firstly processed by Sanger sequencing. If validation results were unavailable or inconsistent with NGS calls, another validation test would be performed by mass spectrometry genotyping. RESULTS:A total of 6,939 high quality NGS variants with ?35 × depth coverage and ?35% heterozygous ratio were 100% confirmed by a secondary methodology. 5,775 heterozygous variants were separated from 760 homozygous variants and 404 hemizygous variants by 80% heterozygous ratio. A total of 1.5% (98/6,939) of NGS variants were validated by mass spectrometry genotyping. CONCLUSION:Considering of the above comprehensive assessment, a new variant with high quality from a well-validated capture-based NGS workflow can be reported directly without validation.

SUBMITTER: Zheng J

PROVIDER: S-EPMC6625156 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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A comprehensive assessment of Next-Generation Sequencing variants validation using a secondary technology.

Zheng Jianchao J Zhang Hongyun H Banerjee Santasree S Li Yun Y Zhou Junyu J Yang Qian Q Tan Xuemei X Han Peng P Fu Qinmei Q Cui Xiaoli X Yuan Yuying Y Zhang Meiyan M Shen Ruiqin R Song Haifeng H Zhang Xiuqing X Zhao Lijian L Peng Zhiyu Z Wang Wei W Yin Ye Y

Molecular genetics & genomic medicine 20190604 7

<h4>Background</h4>Recently, increasing innovations improved the accuracy of next generation sequencing (NGS) data. However, the validation of all NGS variants increased the cost and turn-around time of clinical diagnosis, and therefore limited the further development of clinical applications. We aimed to comprehensively assess the necessity of validating NGS variants.<h4>Methods</h4>Validation data of 7,601 NGS variants involving 1,045 genes were collected from 5,190 clinical samples and sequen ...[more]

PMID: 31165590

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Project description:The impact of natural killer (NK) cell alloreactivity on hematopoietic stem cell transplantation (HSCT) outcome is still debated due to the complexity of graft parameters, HLA class I environment, the nature of killer cell immunoglobulin-like receptor (KIR)/KIR ligand genetic combinations studied, and KIR+ NK cell repertoire size. KIR genes are known to be polymorphic in terms of gene content, copy number variation, and number of alleles. These allelic polymorphisms may impact both the phenotype and function of KIR+ NK cells. We, therefore, speculate that polymorphisms may alter donor KIR+ NK cell phenotype/function thus modulating post-HSCT KIR+ NK cell alloreactivity. To investigate KIR allele polymorphisms of all KIR genes, we developed a next-generation sequencing (NGS) technology on a MiSeq platform. To ensure the reliability and specificity of our method, genomic DNA from well-characterized cell lines were used; high-resolution KIR typing results obtained were then compared to those previously reported. Two different bioinformatic pipelines were used allowing the attribution of sequencing reads to specific KIR genes and the assignment of KIR alleles for each KIR gene. Our results demonstrated successful long-range KIR gene amplifications of all reference samples using intergenic KIR primers. The alignment of reads to the human genome reference (hg19) using BiRD pipeline or visualization of data using Profiler software demonstrated that all KIR genes were completely sequenced with a sufficient read depth (mean 317× for all loci) and a high percentage of mapping (mean 93% for all loci). Comparison of high-resolution KIR typing obtained to those published data using exome capture resulted in a reported concordance rate of 95% for centromeric and telomeric KIR genes. Overall, our results suggest that NGS can be used to investigate the broad KIR allelic polymorphism. Hence, these data improve our knowledge, not only on KIR+ NK cell alloreactivity in HSCT but also on the role of KIR+ NK cell populations in control of viral infections and diseases.

Dataset Information

A comprehensive assessment of Next-Generation Sequencing variants validation using a secondary technology.

Publications

A comprehensive assessment of Next-Generation Sequencing variants validation using a secondary technology.

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