From the Guidelines
Next-generation sequencing (NGS) is a highly effective tool for detecting germline mutations, offering advantages over traditional sequencing methods in terms of higher throughput, sensitivity, and ability to examine multiple genes simultaneously. NGS technologies analyze DNA sequences by breaking them into fragments and reading them simultaneously, allowing for comprehensive examination of an individual's genetic material. When applied to germline mutation detection, NGS can identify inherited genetic variants present in reproductive cells that are passed from parents to offspring. The process typically involves extracting DNA from blood or saliva samples, preparing DNA libraries, sequencing these libraries on NGS platforms, and analyzing the resulting data to identify variants. Some key considerations for germline variant calling include:
- Choosing a single variant caller that meets the needs of the laboratory, such as GATK HaplotypeCaller, which has demonstrated high accuracy (F-scores > 0.99) in numerous benchmark datasets 1
- Combining the results of two orthogonal SNV/indel callers, such as HaplotypeCaller and Platypus, may offer a slight sensitivity advantage 1
- Using software packages such as BCFtools to merge and reconcile multiple variant callsets into one, while taking care to properly handle complex variants and/or differences in variant representation 1 NGS can detect various types of germline mutations, including single nucleotide variants, small insertions and deletions, and copy number variations, making it a valuable tool in medical genetics for hereditary cancer risk assessment, carrier screening for recessive disorders, and diagnosing inherited conditions, as discussed in recent studies 1.
From the Research
Detection of Germline Mutations using Next-Generation Sequencing (NGS)
- Next-generation sequencing (NGS) can detect germline mutations, as evident from several studies 2, 3, 4, 5, 6.
- NGS data may help identify single nucleotide variants, insertions/deletions, copy number variations, and translocations, which can be used to detect germline mutations 2.
- Paired tumor-normal targeted NGS can detect germline variants, including pathogenic germline mutations in DNA mismatch repair (MMR) genes that underlie Lynch syndrome 4.
- Targeted next-generation sequencing (TNGS) can be used to assess the mosaic ratios of known sites and detect germline mosaicism 5.
- NGS can be applied to the whole or to selected parts of the genome, making it a valuable tool for genetic diagnosis, including the detection of germline mutations 6.
Limitations and Challenges
- NGS has limitations, such as coverage and accuracy of <100%, resulting in missing variants and false positive findings 6.
- Variant interpretation is challenging, as there is usually more than one candidate variant found, highlighting the need for standards for NGS with respect to run quality and variant interpretation 6.
- Ethical challenges, including incidental findings and guiding unaffected probands seeking direct-to-customer testing, must be addressed when using NGS for genetic diagnosis 6.
Clinical Applications
- NGS can identify germline mutations, which can help reveal Lynch syndrome in cancer patients and have important implications for cancer screening and risk reduction in these patients and their families 4.
- NGS can be used to detect paternal germline mosaicism, providing a strategy to determine germline mosaicism and inform genetic and reproductive counseling 5.
- The application of NGS in research and diagnostics provides a tremendous opportunity to better serve patients, minimizing the "diagnostic odyssey" for patients with genetic disorders 6.