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. 2011 Aug 11;12(8):R73.
doi: 10.1186/gb-2011-12-8-r73.

Hybrid selection for sequencing pathogen genomes from clinical samples

Alexandre Melnikov  1 , Kevin Galinsky, Peter Rogov, Timothy Fennell, Daria Van Tyne, Carsten Russ, Rachel Daniels, Kayla G Barnes, James Bochicchio, Daouda Ndiaye, Papa D Sene, Dyann F Wirth, Chad Nusbaum, Sarah K Volkman, Bruce W Birren, Andreas Gnirke, Daniel E Neafsey
Affiliations

Hybrid selection for sequencing pathogen genomes from clinical samples

Alexandre Melnikov et al. Genome Biol. .

Abstract

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla.

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Figures

Figure 1
Figure 1
Sequencing coverage plots from a randomly chosen region of P. falciparum chromosome 1. (a) Unamplified (green) and WGA (purple) WGBs compared to pure P. falciparum (gray). (b) Unamplified (green) and WGA (purple) synthetic bait read coverage compared to pure P. falciparum (gray). Red bars indicate bait locations. (c) Local %GC (the percentage of nucleotides in the genome that are G or C; in 140-bp windows). Green bars indicate exons. Chr, chromosome.
Figure 2
Figure 2
Genome-wide sequencing coverage and composition. (a) Coverage thresholds for unamplified (green) and WGA (purple) whole genome baits compared to pure P. falciparum (gray) and simulated coverage from a non-hybrid-selected mock clinical sample (yellow). (b) Genome-wide coverage as a function of %GC. The vertical black line represents average exonic %GC. The red histogram represents the probability density distribution of genome composition (right vertical axis). Lines depict coverage (left vertical axis) of pure P. falciparum DNA (gray), as well as unamplified (green) and WGA (purple) hybrid-selected samples initially containing 1% P. falciparum DNA. %GC, the percentage of nucleotides in the genome which are G or C.
Figure 3
Figure 3
Principal component analysis plot based on SNP calls produced from hybrid-selected and non-hybrid-selected samples. The hybrid-selected clinical sample from Senegal (red) clusters with 12 previously sequenced Senegal samples (blue). The hybrid-selected 3D7 samples (red) cluster with the non-hybrid-selected 3D7 sample (yellow). P. falciparum isolates from India (purple) and Thailand (brown) are also represented. PC, principal content.
See this image and copyright information in PMC

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