17 research outputs found
Comparing Three Approaches
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 Āµg 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 sample
Exome Sequencing from Nanogram Amounts of Starting DNA: Comparing Three Approaches
<div><p>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 SureSelect<i><sup>XT2</sup></i> Human AllExon v4+UTRs capture probes, and HiSeq2000 sequencing were performed for test libraries along with the control library prepared from 1 Āµg 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.</p></div
The experimental scheme.
<p>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.</p
Coverage statistics for the target region.
<p>Analysis was performed on subsets of reads uniquely mapped to the target region and having approximately equal total amounts of bases (<b>ā¼</b>17<b>Ć</b>10<sup>8</sup> bases).</p
Per-base sequencing depth distribution on the target region.
<p>Per-base sequencing depth distribution on the target region.</p
Alignment statistics.
<p>*high confident reads-reads with probability of wrong mapping lower than 0.05 according to their MAPQ score (MAPQ>13).</p><p>**some of GenomePlex ES library reads contained sequences of the primer used for whole genome amplification. These common segments were cut out before the alignment. As a result, 13.8% of and 11.9% of nucleotides were removed from the reads of the Test DNA 1 and Test DNA 2 libraries, respectively.</p><p>***FR-flanking regions (FR), which include 100<b> </b>bp from both ends of the targeted sequences.</p
Profiles of coverage depth along the target region for Test DNA 1 (upper panel) and Test DNA 2 (lower panel) WES libraries.
<p>Profiles of coverage depth along the target region for Test DNA 1 (upper panel) and Test DNA 2 (lower panel) WES libraries.</p
Sharing of genetic variations between strategies depicted in a Venn diagram.
<p>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 ESā=āSt; ThruPLEX-FD ESā=āTp; REPLI-g ESā=āRg; GenomePlex ESā=āGp. The lower left tile presents the overall statistics, where āTotalā indicates the number of all unique SNVs found in the region of interest, i.e. the union of SNV sets found by each strategy.</p
Coverage distribution along the target regions with different percentages of GC bases.
<p>Coverage distribution along the target regions with different percentages of GC bases.</p