Smart Nonuniformity: Calibration of Sequencing Depth of a Targeted Gene Panel to Simultaneously Detect Somatic and Germline Variants.

IF 3.4 3区 医学 Q1 PATHOLOGY
Robert L O'Reilly, Philip Harraka, Jared Burke, Daniele Belluoccio, Paul Yeh, Kerryn Howlett, Kiarash Behrouzfar, Amanda Rewse, Helen Tsimiklis, Graham G Giles, John L Hopper, Kristen J Bubb, Stephen J Nicholls, Roger L Milne, Melissa C Southey
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引用次数: 0

Abstract

Targeted gene panel sequencing that measures genomic variation at different depths has potential diagnostic application. A targeted gene panel was developed to detect somatic variants associated with clonal hematopoiesis of indeterminate potential (CHIP), which requires an optimal sequencing depth of >500×; and germline variants requiring a lower ≥50× depth (panel 1). This was achieved by adjusting probe ratios for genomic regions relevant to identifying CHIP in comparison to those relevant to germline variation analysis. An additional custom panel containing only the genomic regions relevant to the identification of CHIP (panel 2) was also manufactured to confirm that panel 1 did not miss variants because of the complex design. Both panels were used to sequence 150 blood-derived DNAs; 94 DNAs from research participants aged 64 to 75 years; 16 DNAs with known germline variants; 16 DNAs with known germline variants (titrated from 0% to 100%); 24 DNAs from individuals aged <40 years; and 3 commercial CHIP controls and 3 high-molecular-weight DNA controls. The sequencing median depth ratio between the CHIP and germline relevant genomic regions was 4.7:1. Fourteen CHIP-associated variants were called in both panel 1 (1382× median variant depth) and panel 2 (1665× median variant depth). All known germline variants were identified (251× median variant depth). Smart nonuniformity sequencing reliably detects variants with allele frequency in the range >0.01 to 1 in one workflow.

智能非均匀性:校准目标基因面板的测序深度,同时检测体细胞和种系变异。
在不同深度测量基因组变异的靶向基因面板测序具有潜在的诊断应用。开发了一个靶向基因面板,用于检测1)与不确定潜力克隆造血(CHIP)相关的体细胞变异,这需要bb0 500X的最佳测序深度,以及2)需要更低的≥50X深度的种系变异[面板1]。这是通过调整与鉴定CHIP相关的基因组区域的探针比例与与种系变异分析相关的探针比例来实现的。还制作了一个额外的定制面板,仅包含与CHIP鉴定相关的基因组区域[面板2],以确认面板1不会因复杂的设计而遗漏变异。两个面板都用于对150个血液来源的dna进行测序;来自64-75岁研究参与者的94个dna;16个已知种系变异的dna;16个已知种系变异的dna(从0-100%滴定);在一个工作流程中,来自0.01-1岁个体的24个dna。
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来源期刊
CiteScore
8.10
自引率
2.40%
发文量
143
审稿时长
43 days
期刊介绍: The Journal of Molecular Diagnostics, the official publication of the Association for Molecular Pathology (AMP), co-owned by the American Society for Investigative Pathology (ASIP), seeks to publish high quality original papers on scientific advances in the translation and validation of molecular discoveries in medicine into the clinical diagnostic setting, and the description and application of technological advances in the field of molecular diagnostic medicine. The editors welcome for review articles that contain: novel discoveries or clinicopathologic correlations including studies in oncology, infectious diseases, inherited diseases, predisposition to disease, clinical informatics, or the description of polymorphisms linked to disease states or normal variations; the application of diagnostic methodologies in clinical trials; or the development of new or improved molecular methods which may be applied to diagnosis or monitoring of disease or disease predisposition.
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