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CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing
A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8031414/ https://www.ncbi.nlm.nih.gov/pubmed/33830997 http://dx.doi.org/10.1371/journal.pone.0241253 |
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author | Wallace, Amelia D. Sasani, Thomas A. Swanier, Jordan Gates, Brooke L. Greenland, Jeff Pedersen, Brent S. Varley, Katherine E. Quinlan, Aaron R. |
author_facet | Wallace, Amelia D. Sasani, Thomas A. Swanier, Jordan Gates, Brooke L. Greenland, Jeff Pedersen, Brent S. Varley, Katherine E. Quinlan, Aaron R. |
author_sort | Wallace, Amelia D. |
collection | PubMed |
description | A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore’s MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39–416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease. |
format | Online Article Text |
id | pubmed-8031414 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-80314142021-04-14 CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing Wallace, Amelia D. Sasani, Thomas A. Swanier, Jordan Gates, Brooke L. Greenland, Jeff Pedersen, Brent S. Varley, Katherine E. Quinlan, Aaron R. PLoS One Research Article A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore’s MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39–416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease. Public Library of Science 2021-04-08 /pmc/articles/PMC8031414/ /pubmed/33830997 http://dx.doi.org/10.1371/journal.pone.0241253 Text en © 2021 Wallace et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Wallace, Amelia D. Sasani, Thomas A. Swanier, Jordan Gates, Brooke L. Greenland, Jeff Pedersen, Brent S. Varley, Katherine E. Quinlan, Aaron R. CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing |
title | CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing |
title_full | CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing |
title_fullStr | CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing |
title_full_unstemmed | CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing |
title_short | CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing |
title_sort | cabage: a cas9-based background elimination strategy for targeted, long-read dna sequencing |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8031414/ https://www.ncbi.nlm.nih.gov/pubmed/33830997 http://dx.doi.org/10.1371/journal.pone.0241253 |
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