Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing
Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine–cytosine (GC) content, including repeat expansions associated with human...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2018
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175010/ https://www.ncbi.nlm.nih.gov/pubmed/29932473 http://dx.doi.org/10.1002/humu.23580 |
_version_ | 1783361408715456512 |
---|---|
author | Höijer, Ida Tsai, Yu‐Chih Clark, Tyson A. Kotturi, Paul Dahl, Niklas Stattin, Eva‐Lena Bondeson, Marie‐Louise Feuk, Lars Gyllensten, Ulf Ameur, Adam |
author_facet | Höijer, Ida Tsai, Yu‐Chih Clark, Tyson A. Kotturi, Paul Dahl, Niklas Stattin, Eva‐Lena Bondeson, Marie‐Louise Feuk, Lars Gyllensten, Ulf Ameur, Adam |
author_sort | Höijer, Ida |
collection | PubMed |
description | Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine–cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification‐free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No‐Amp Targeted sequencing) in combination with single molecule, real‐time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification‐free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No‐Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR‐based methods. |
format | Online Article Text |
id | pubmed-6175010 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-61750102018-10-15 Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing Höijer, Ida Tsai, Yu‐Chih Clark, Tyson A. Kotturi, Paul Dahl, Niklas Stattin, Eva‐Lena Bondeson, Marie‐Louise Feuk, Lars Gyllensten, Ulf Ameur, Adam Hum Mutat Research Articles Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine–cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification‐free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No‐Amp Targeted sequencing) in combination with single molecule, real‐time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification‐free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No‐Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR‐based methods. John Wiley and Sons Inc. 2018-07-12 2018-09 /pmc/articles/PMC6175010/ /pubmed/29932473 http://dx.doi.org/10.1002/humu.23580 Text en © 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Höijer, Ida Tsai, Yu‐Chih Clark, Tyson A. Kotturi, Paul Dahl, Niklas Stattin, Eva‐Lena Bondeson, Marie‐Louise Feuk, Lars Gyllensten, Ulf Ameur, Adam Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing |
title | Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing |
title_full | Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing |
title_fullStr | Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing |
title_full_unstemmed | Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing |
title_short | Detailed analysis of HTT repeat elements in human blood using targeted amplification‐free long‐read sequencing |
title_sort | detailed analysis of htt repeat elements in human blood using targeted amplification‐free long‐read sequencing |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175010/ https://www.ncbi.nlm.nih.gov/pubmed/29932473 http://dx.doi.org/10.1002/humu.23580 |
work_keys_str_mv | AT hoijerida detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT tsaiyuchih detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT clarktysona detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT kotturipaul detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT dahlniklas detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT stattinevalena detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT bondesonmarielouise detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT feuklars detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT gyllenstenulf detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing AT ameuradam detailedanalysisofhttrepeatelementsinhumanbloodusingtargetedamplificationfreelongreadsequencing |