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Comparative Study
. 2014 Jul 3;9(7):e101154.
doi: 10.1371/journal.pone.0101154. eCollection 2014.

Exome sequencing from nanogram amounts of starting DNA: comparing three approaches

Vera N Rykalina  1 , Alexey A Shadrin  2 , Vyacheslav S Amstislavskiy  3 , Evgeny I Rogaev  4 , Hans Lehrach  3 , Tatiana A Borodina  5
Affiliations
Comparative Study

Exome sequencing from nanogram amounts of starting DNA: comparing three approaches

Vera N Rykalina et al. PLoS One. .

Abstract

Hybridization-based target enrichment protocols require relatively large starting amounts of genomic DNA, which is not always available. Here, we tested three approaches to pre-capture library preparation starting from 10 ng of genomic DNA: (i and ii) whole-genome amplification of DNA samples with REPLI-g (Qiagen) and GenomePlex (Sigma) kits followed by standard library preparation, and (iii) library construction with a low input oriented ThruPLEX kit (Rubicon Genomics). Exome capture with Agilent SureSelectXT2 Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 µg of starting DNA. Tested protocols were characterized in terms of mapping efficiency, enrichment ratio, coverage of the target region, and reliability of SNP genotyping. REPLI-g- and ThruPLEX-FD-based protocols seem to be adequate solutions for exome sequencing of low input samples.

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Conflict of interest statement

Competing Interests: The authors declare that no competing interests exist. Though two authors (VR and TB) are currently employed by a commercial company Alacris Theranostics GmbH, this does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials. Results presented in the current manuscript are not IP of the company and have no relationship to the research performed at Alacris Theranostics GmbH or to the services provided by this company. The presence of the company’s name in the list of affiliations merely reflects the fact that during the preparation of the manuscript affiliations of two of the authors have changed.

Figures

Figure 1
Figure 1. The experimental scheme.
Two DNA samples (Test DNA 1 and Test DNA 2) were subjected to four exome sequencing (ES) protocols performed in parallel: control (Standard ES) and three modified (REPLI-g ES, GenomePlex ES and ThruPLEX-FD ES). Common steps performed in parallel for several protocols are shown by text boxes spanning the corresponding number of protocol columns.
Figure 2
Figure 2. Per-base sequencing depth distribution on the target region.
Figure 3
Figure 3. Coverage distribution along the target regions with different percentages of GC bases.
Figure 4
Figure 4. Profiles of coverage depth along the target region for Test DNA 1 (upper panel) and Test DNA 2 (lower panel) WES libraries.
Figure 5
Figure 5. Sharing of genetic variations between strategies depicted in a Venn diagram.
Only variation with minimum depth of coverage of 20x and minimum quality of 13 were taken into account in all four strategies. The names of the samples are abbreviated: Standard ES = St; ThruPLEX-FD ES = Tp; REPLI-g ES = Rg; GenomePlex ES = Gp. The lower left tile presents the overall statistics, where “Total” indicates the number of all unique SNVs found in the region of interest, i.e. the union of SNV sets found by each strategy.
See this image and copyright information in PMC

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