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PLoS One. 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.

Author information

1
Max-Planck Institute for Molecular Genetics, Berlin, Germany; AlacrisTheranostics GmbH, Berlin, Germany; Freie Universität Berlin, Berlin, Germany.
2
Max-Planck Institute for Molecular Genetics, Berlin, Germany; Freie Universität Berlin, Berlin, Germany.
3
Max-Planck Institute for Molecular Genetics, Berlin, Germany.
4
Vavilov Institute of General Genetics, Moscow, Russia.
5
Max-Planck Institute for Molecular Genetics, Berlin, Germany; AlacrisTheranostics GmbH, Berlin, Germany.

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.

PMID:
24992588
PMCID:
PMC4081514
DOI:
10.1371/journal.pone.0101154
[Indexed for MEDLINE]
Free PMC Article

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