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Title: An in-solution hybridisation method for the isolation of pathogen DNA from human DNA-rich clinical samples for analysis by NGS
Authors: Miriam Smith
Susana Campino
Yong Gu
Taane G. Clark
Thomas D. Otto
Gareth Maslen
Magnus Manske
Mallika Imwong
Arjen M. Dondorp
Dominic P. Kwiatkowski
Michael A. Quail
Harold Swerdlow
Wellcome Trust Sanger Institute
London School of Hygiene & Tropical Medicine
Mahidol University
Churchill Hospital
University of Oxford
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 11-Jun-2012
Citation: Open Genomics Journal. Vol.5, No.1 (2012), 18-29
Abstract: Studies on DNA from pathogenic organisms, within clinical samples, are often complicated by the presence of large amounts of host, e.g., human DNA. Isolation of pathogen DNA from these samples would improve the efficiency of next-generation sequencing (NGS) and pathogen identification. Here we describe a solution-based hybridisation method for isolation of pathogen DNA from a mixed population. This straightforward and inexpensive technique uses probes made from whole-genome DNA and off-the-shelf reagents. In this study, Escherichia coli DNA was successfully enriched from a mixture of E.coli and human DNA. After enrichment, genome coverage following NGS was significantly higher and the evenness of coverage and GC content were unaffected. This technique was also applied to samples containing a mixture of human and Plasmodium falciparum DNA. The P.falciparum genome is particularly difficult to sequence due to its high AT content (80.6%) and repetitive nature. Post enrichment, a bias in the recovered DNA was observed, with a poorer representation of the AT-rich non-coding regions. This uneven coverage was also observed in pre-enrichment samples, but to a lesser degree. Despite the coverage bias in enriched samples, SNP (single-nucleotide polymorphism) calling in coding regions was unaffected and the majority of samples had over 90% of their coding region covered at 5x depth. This technique shows significant promise as an effective method to enrich pathogen DNA from samples with heavy human contamination, particularly when applied to GC-neutral genomes. © Smith et al.
ISSN: 1875693X
Appears in Collections:Scopus 2011-2015

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