Nanopore adaptive sampling accurately detects nucleotide variants and improves the characterization of large-scale rearrangement for the diagnosis of cancer predisposition

IF 7.9 1区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL

Clinical and Translational Medicine Pub Date : 2025-01-09 DOI:10.1002/ctm2.70138

Sandy Chevrier, Corentin Richard, Marie Mille, Denis Bertrand, Romain Boidot

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引用次数: 0

Abstract

Background

Molecular diagnosis has become highly significant for patient management in oncology.

Methods

Here, 30 well-characterized clinical germline samples were studied with adaptive sampling to enrich the full sequence of 152 cancer predisposition genes. Sequencing was performed on Oxford Nanopore (ONT) R10.4.1 MinION flowcells with the Q20+ chemistry.

Results

In our cohort, 11 samples had large-scale rearrangements (LSR), which were all detected with ONT sequencing. In addition to perfectly detecting the locus of the LSR, we found a known MLPA amplification of exon 13 in the BRCA1 (NM_7294) gene corresponded to a duplication in tandem of both exons 12 and 13 of the reference NM_7300. Similarly, in another sample with a known total deletion of the BRCA1 gene, ONT sequencing highlighted this complete deletion was the consequence of a large deletion of almost 140 000 bp carrying over five different genes. ONT sequencing was also able to detect all pathogenic nucleotide variants present in 16 samples at low coverage. As we analyzed complete genes and more genes than with short-read sequencing, we detected novel unknown variants. We randomly selected six new variants with a coverage larger than 10× and an average quality higher than 14, and confirmed all of them by Sanger sequencing, suggesting that variants detected with ONT (coverage >10× and quality score >14) could be considered as real variants.

Conclusions

We showed that ONT adaptive sampling sequencing is suitable for the analysis of germline alterations, improves characterization of LSR, and detects single nucleotide variations even at low coverage.

Key points

Adaptive sampling is suitable for the analysis of germline alterations.
Improves the characterization of Large Scale Rearrangement and detects SNV at a minimum coverage of 10x.
Allows flexibility of sequencing.

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查看原文本刊更多论文

纳米孔自适应采样准确地检测核苷酸变异，并提高了癌症易感性诊断的大规模重排特征。

背景：分子诊断在肿瘤患者管理中已经变得非常重要。方法：本研究采用自适应取样方法对30例具有良好特征的临床种系样本进行研究，以丰富152个癌症易感基因的全序列。采用Q20+化学方法对Oxford Nanopore (ONT) R10.4.1 MinION流式细胞进行测序。结果：在我们的队列中，11个样本存在大规模重排（LSR），均通过ONT测序检测到。除了完美地检测到LSR位点外，我们还发现BRCA1 （NM_7294）基因外显子13的已知MLPA扩增与参考基因NM_7300的外显子12和13的串联重复相对应。同样，在另一个已知BRCA1基因完全缺失的样本中，ONT测序强调了这种完全缺失是携带5种不同基因的近140,000 bp的大缺失的结果。ONT测序还能够在低覆盖率下检测到16个样本中存在的所有致病性核苷酸变异。当我们分析完整的基因和比短读测序更多的基因时，我们发现了新的未知变异。我们随机选择了6个覆盖率大于10倍、平均质量大于14的新变异，并通过Sanger测序对其进行了确认，表明ONT检测到的变异（覆盖率为10倍，质量评分为14分）可以被认为是真正的变异。结论：我们发现ONT自适应采样测序适用于种系改变的分析，提高了LSR的表征，即使在低覆盖率下也能检测到单核苷酸变异。重点：自适应取样适用于种系改变的分析。改进了大规模重排的特性，并在最小覆盖率为10倍的情况下检测SNV。允许排序的灵活性。

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来源期刊

Clinical and Translational Medicine Multiple-

CiteScore

15.90

自引率

1.90%

发文量

450

审稿时长

4 weeks

期刊介绍： Clinical and Translational Medicine (CTM) is an international, peer-reviewed, open-access journal dedicated to accelerating the translation of preclinical research into clinical applications and fostering communication between basic and clinical scientists. It highlights the clinical potential and application of various fields including biotechnologies, biomaterials, bioengineering, biomarkers, molecular medicine, omics science, bioinformatics, immunology, molecular imaging, drug discovery, regulation, and health policy. With a focus on the bench-to-bedside approach, CTM prioritizes studies and clinical observations that generate hypotheses relevant to patients and diseases, guiding investigations in cellular and molecular medicine. The journal encourages submissions from clinicians, researchers, policymakers, and industry professionals.