Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers.
Targeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0208283 |
| _version_ | 1818925320898609152 |
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| author | Joel Pel Amy Leung Wendy W Y Choi Milenko Despotovic W Lloyd Ung Gosuke Shibahara Laura Gelinas Andre Marziali |
| author_facet | Joel Pel Amy Leung Wendy W Y Choi Milenko Despotovic W Lloyd Ung Gosuke Shibahara Laura Gelinas Andre Marziali |
| author_sort | Joel Pel |
| collection | DOAJ |
| description | Targeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost. We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined 'target-capture-PCR' workflow. This approach uses physically linked capture probes and PCR primers and is expected to work with panel sizes from 100 bp to >10 Mbp. It reduces the time and complexity of the capture workflow, eliminates long hybridization and wash steps and enables rapid library construction and target capture. High on-target read fractions are achievable due to repeated sequence selection in the target-capture-PCR step, thus lowering sequencing cost. We have demonstrated this technology on sample types including cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) derived DNA, capturing a 35-gene pan-cancer panel, and therein detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), variants as low as 0.25% abundance were detected, limited by input mass and sequencing depth. Additionally, sequencing libraries were prepared in less than eight hours from extracted DNA to loaded sequencer, demonstrating that LTC holds promise as a broadly applicable tool for rapid, cost-effective and high performance targeted sequencing. |
| first_indexed | 2024-12-20T02:39:21Z |
| format | Article |
| id | doaj.art-601b93ed87364a7e9d2abbbdcb593b4f |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T02:39:21Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-601b93ed87364a7e9d2abbbdcb593b4f2022-12-21T19:56:21ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011312e020828310.1371/journal.pone.0208283Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers.Joel PelAmy LeungWendy W Y ChoiMilenko DespotovicW Lloyd UngGosuke ShibaharaLaura GelinasAndre MarzialiTargeted Next Generation Sequencing (NGS) is being adopted increasingly broadly in many research, commercial and clinical settings. Currently used target capture methods, however, typically require complex and lengthy (sometimes multi-day) workflows that complicates their use in certain applications. In addition, small panels for high sequencing depth applications such as liquid biopsy typically have low on-target rates, resulting in unnecessarily high sequencing cost. We have developed a novel targeted sequencing library preparation method, named Linked Target Capture (LTC), which replaces typical multi-day target capture workflows with a single-day, combined 'target-capture-PCR' workflow. This approach uses physically linked capture probes and PCR primers and is expected to work with panel sizes from 100 bp to >10 Mbp. It reduces the time and complexity of the capture workflow, eliminates long hybridization and wash steps and enables rapid library construction and target capture. High on-target read fractions are achievable due to repeated sequence selection in the target-capture-PCR step, thus lowering sequencing cost. We have demonstrated this technology on sample types including cell-free DNA (cfDNA) and formalin-fixed, paraffin-embedded (FFPE) derived DNA, capturing a 35-gene pan-cancer panel, and therein detecting single nucleotide variants, copy number variants, insertions, deletions and gene fusions. With the integration of unique molecular identifiers (UMIs), variants as low as 0.25% abundance were detected, limited by input mass and sequencing depth. Additionally, sequencing libraries were prepared in less than eight hours from extracted DNA to loaded sequencer, demonstrating that LTC holds promise as a broadly applicable tool for rapid, cost-effective and high performance targeted sequencing.https://doi.org/10.1371/journal.pone.0208283 |
| spellingShingle | Joel Pel Amy Leung Wendy W Y Choi Milenko Despotovic W Lloyd Ung Gosuke Shibahara Laura Gelinas Andre Marziali Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers. PLoS ONE |
| title | Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers. |
| title_full | Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers. |
| title_fullStr | Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers. |
| title_full_unstemmed | Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers. |
| title_short | Rapid and highly-specific generation of targeted DNA sequencing libraries enabled by linking capture probes with universal primers. |
| title_sort | rapid and highly specific generation of targeted dna sequencing libraries enabled by linking capture probes with universal primers |
| url | https://doi.org/10.1371/journal.pone.0208283 |
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