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SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing

Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously ana...

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Autores principales: Guo, Xiaoge, Lehner, Kevin, O’Connell, Karen, Zhang, Jenny, Dave, Sandeep S., Jinks-Robertson, Sue
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683651/
https://www.ncbi.nlm.nih.gov/pubmed/26497143
http://dx.doi.org/10.1534/g3.115.023317
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author Guo, Xiaoge
Lehner, Kevin
O’Connell, Karen
Zhang, Jenny
Dave, Sandeep S.
Jinks-Robertson, Sue
author_facet Guo, Xiaoge
Lehner, Kevin
O’Connell, Karen
Zhang, Jenny
Dave, Sandeep S.
Jinks-Robertson, Sue
author_sort Guo, Xiaoge
collection PubMed
description Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously analyze one or a small number of genomic targets derived from multiple sources. In the budding yeast system, SMRT sequencing was used to analyze strand-exchange intermediates generated during mitotic recombination and to analyze genetic changes in a forward mutation assay. The general barcoding-SMRT approach was then extended to diffuse large B-cell lymphoma primary tumors and cell lines, where detected changes agreed with prior Illumina exome sequencing. A distinct advantage afforded by SMRT sequencing over other next-gen methods is that it immediately provides the linkage relationships between SNPs in the target segment sequenced. The strength of our approach for mutation/recombination studies (as well as linkage identification) derives from its inherent computational simplicity coupled with a lack of reliance on sophisticated statistical analyses.
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spelling pubmed-46836512015-12-18 SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing Guo, Xiaoge Lehner, Kevin O’Connell, Karen Zhang, Jenny Dave, Sandeep S. Jinks-Robertson, Sue G3 (Bethesda) Investigations Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously analyze one or a small number of genomic targets derived from multiple sources. In the budding yeast system, SMRT sequencing was used to analyze strand-exchange intermediates generated during mitotic recombination and to analyze genetic changes in a forward mutation assay. The general barcoding-SMRT approach was then extended to diffuse large B-cell lymphoma primary tumors and cell lines, where detected changes agreed with prior Illumina exome sequencing. A distinct advantage afforded by SMRT sequencing over other next-gen methods is that it immediately provides the linkage relationships between SNPs in the target segment sequenced. The strength of our approach for mutation/recombination studies (as well as linkage identification) derives from its inherent computational simplicity coupled with a lack of reliance on sophisticated statistical analyses. Genetics Society of America 2015-10-22 /pmc/articles/PMC4683651/ /pubmed/26497143 http://dx.doi.org/10.1534/g3.115.023317 Text en Copyright © 2015 Guo et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Guo, Xiaoge
Lehner, Kevin
O’Connell, Karen
Zhang, Jenny
Dave, Sandeep S.
Jinks-Robertson, Sue
SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
title SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
title_full SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
title_fullStr SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
title_full_unstemmed SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
title_short SMRT Sequencing for Parallel Analysis of Multiple Targets and Accurate SNP Phasing
title_sort smrt sequencing for parallel analysis of multiple targets and accurate snp phasing
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683651/
https://www.ncbi.nlm.nih.gov/pubmed/26497143
http://dx.doi.org/10.1534/g3.115.023317
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