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Enhanced whole exome sequencing by higher DNA insert lengths

BACKGROUND: Whole exome sequencing (WES) has been proven to serve as a valuable basis for various applications such as variant calling and copy number variation (CNV) analyses. For those analyses the read coverage should be optimally balanced throughout protein coding regions at sufficient read dept...

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Autores principales: Pommerenke, Claudia, Geffers, Robert, Bunk, Boyke, Bhuju, Sabin, Eberth, Sonja, Drexler, Hans G., Quentmeier, Hilmar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880973/
https://www.ncbi.nlm.nih.gov/pubmed/27225215
http://dx.doi.org/10.1186/s12864-016-2698-y
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author Pommerenke, Claudia
Geffers, Robert
Bunk, Boyke
Bhuju, Sabin
Eberth, Sonja
Drexler, Hans G.
Quentmeier, Hilmar
author_facet Pommerenke, Claudia
Geffers, Robert
Bunk, Boyke
Bhuju, Sabin
Eberth, Sonja
Drexler, Hans G.
Quentmeier, Hilmar
author_sort Pommerenke, Claudia
collection PubMed
description BACKGROUND: Whole exome sequencing (WES) has been proven to serve as a valuable basis for various applications such as variant calling and copy number variation (CNV) analyses. For those analyses the read coverage should be optimally balanced throughout protein coding regions at sufficient read depth. Unfortunately, WES is known for its uneven coverage within coding regions due to GC-rich regions or off-target enrichment. RESULTS: In order to examine the irregularities of WES within genes, we applied Agilent SureSelectXT exome capture on human samples and sequenced these via Illumina in 2 × 101 paired-end mode. As we suspected the sequenced insert length to be crucial in the uneven coverage of exome captured samples, we sheared 12 genomic DNA samples to two different DNA insert size lengths, namely 130 and 170 bp. Interestingly, although mean coverages of target regions were clearly higher in samples of 130 bp insert length, the level of evenness was more pronounced in 170 bp samples. Moreover, merging overlapping paired-end reads revealed a positive effect on evenness indicating overlapping reads as another reason for the unevenness. In addition, mutation analysis on a subset of the samples was performed. In these isogenic subclones, the false negative rate in the 130 bp samples was almost double to that in the 170 bp samples. Visual inspection of the discarded mutation sites exposed low coverages at the sites flanked by high amplitudes of coverage depth. CONCLUSIONS: Producing longer insert reads could be a good strategy to achieve better uniform read coverage in coding regions and hereby enhancing the effective sequencing yield to provide an improved basis for further variant calling and CNV analyses.
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spelling pubmed-48809732016-05-27 Enhanced whole exome sequencing by higher DNA insert lengths Pommerenke, Claudia Geffers, Robert Bunk, Boyke Bhuju, Sabin Eberth, Sonja Drexler, Hans G. Quentmeier, Hilmar BMC Genomics Methodology Article BACKGROUND: Whole exome sequencing (WES) has been proven to serve as a valuable basis for various applications such as variant calling and copy number variation (CNV) analyses. For those analyses the read coverage should be optimally balanced throughout protein coding regions at sufficient read depth. Unfortunately, WES is known for its uneven coverage within coding regions due to GC-rich regions or off-target enrichment. RESULTS: In order to examine the irregularities of WES within genes, we applied Agilent SureSelectXT exome capture on human samples and sequenced these via Illumina in 2 × 101 paired-end mode. As we suspected the sequenced insert length to be crucial in the uneven coverage of exome captured samples, we sheared 12 genomic DNA samples to two different DNA insert size lengths, namely 130 and 170 bp. Interestingly, although mean coverages of target regions were clearly higher in samples of 130 bp insert length, the level of evenness was more pronounced in 170 bp samples. Moreover, merging overlapping paired-end reads revealed a positive effect on evenness indicating overlapping reads as another reason for the unevenness. In addition, mutation analysis on a subset of the samples was performed. In these isogenic subclones, the false negative rate in the 130 bp samples was almost double to that in the 170 bp samples. Visual inspection of the discarded mutation sites exposed low coverages at the sites flanked by high amplitudes of coverage depth. CONCLUSIONS: Producing longer insert reads could be a good strategy to achieve better uniform read coverage in coding regions and hereby enhancing the effective sequencing yield to provide an improved basis for further variant calling and CNV analyses. BioMed Central 2016-05-25 /pmc/articles/PMC4880973/ /pubmed/27225215 http://dx.doi.org/10.1186/s12864-016-2698-y Text en © Pommerenke et al. 2016 Open Access This article is 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 you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Pommerenke, Claudia
Geffers, Robert
Bunk, Boyke
Bhuju, Sabin
Eberth, Sonja
Drexler, Hans G.
Quentmeier, Hilmar
Enhanced whole exome sequencing by higher DNA insert lengths
title Enhanced whole exome sequencing by higher DNA insert lengths
title_full Enhanced whole exome sequencing by higher DNA insert lengths
title_fullStr Enhanced whole exome sequencing by higher DNA insert lengths
title_full_unstemmed Enhanced whole exome sequencing by higher DNA insert lengths
title_short Enhanced whole exome sequencing by higher DNA insert lengths
title_sort enhanced whole exome sequencing by higher dna insert lengths
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4880973/
https://www.ncbi.nlm.nih.gov/pubmed/27225215
http://dx.doi.org/10.1186/s12864-016-2698-y
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