Format

Send to

Choose Destination
See comment in PubMed Commons below
BMC Genomics. 2016 May 25;17:399. doi: 10.1186/s12864-016-2698-y.

Enhanced whole exome sequencing by higher DNA insert lengths.

Author information

  • 1Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, Braunschweig, 38124, Germany. claudia.pommerenke@dsmz.de.
  • 2Helmholtz Centre for Infection Research, Inhoffenstrasse 7, Braunschweig, 38124, Germany.
  • 3Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, Braunschweig, 38124, Germany.

Abstract

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.

KEYWORDS:

DNA insert size; Evenness score; Read coverage; Variant calling; Whole exome sequencing

PMID:
27225215
PMCID:
PMC4880973
DOI:
10.1186/s12864-016-2698-y
[PubMed - in process]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for PubMed Central
    Loading ...
    Support Center