A robust targeted sequencing approach for low input and variable quality DNA from clinical samples
| Autor: | Ryan T. Koehler, Federico Goodsaid, Jason Stein, Francisco M. De La Vega, Yosr Bouhlal, Susan M. Grimes, Austin P. So, Janet S. Ziegle, Hanlee P. Ji, Daniel Mendoza, Michael Y. Lucero, Yannick Pouliot, Anna Vilborg |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
lcsh:QH426-470 lcsh:Medicine Genomics Computational biology Biology Deep sequencing Article law.invention Exon 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine law Genetics Molecular Biology Gene Genetics (clinical) Polymerase chain reaction 030304 developmental biology Sequence (medicine) 0303 health sciences Oligonucleotide lcsh:R Multiple displacement amplification 3. Good health lcsh:Genetics 030104 developmental biology chemistry 030220 oncology & carcinogenesis Primer (molecular biology) Ligation DNA |
| Zdroj: | NPJ Genomic Medicine npj Genomic Medicine, Vol 3, Iss 1, Pp 1-10 (2018) |
| ISSN: | 2056-7944 |
| Popis: | Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification—this facilitates the accurate assessment of copy number alterations in addition to delivering accurate single nucleotide variant and insertion/deletion detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We demonstrate the performance using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types. Cancer diagnostics: Targeted DNA sequencing for low-quality tumor samples A new DNA sequencing technology enables comprehensive genetic analyses of poor-quality tumor samples. Hanlee Ji from Stanford University in California, USA, together with colleagues from a company he cofounded called TOMA Biosciences, tested the performance of a targeted sequencing assay known as oligonucleotide-selective sequencing (OS-Seq). They used the “in-solution” version of OS-Seq, which involves a pre-processing step to remove any damaged DNA and then sequences target regions of the genome to look for duplications, insertions or deletions of DNA segments. Using archival specimens (which often contain low quantities of degraded DNA) from patients with lung and colorectal cancer, the researchers showed they could detect sequence variants in a panel of 130 cancer-related genes. The findings suggest the OS-Seq assay could help inform treatment decisions for cancer patients, even with clinical specimens of low quality. |
| Databáze: | OpenAIRE |
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