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Open Access Highly Accessed Methodology article

High-throughput 454 resequencing for allele discovery and recombination mapping in Plasmodium falciparum

Upeka Samarakoon1, Allison Regier12, Asako Tan1, Brian A Desany3, Brendan Collins1, John C Tan1, Scott J Emrich12 and Michael T Ferdig1*

Author Affiliations

1 Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame, Notre Dame, IN 46556, USA

2 Department of Computer Science and Engineering, University of Notre Dame, Notre Dame, IN 46556, USA

3 454 Life Sciences, a Roche company, 15 Commercial Street, Branford, CT 06405, USA

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BMC Genomics 2011, 12:116  doi:10.1186/1471-2164-12-116

Published: 17 February 2011

Abstract

Background

Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; however the 81% (A+T) genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites.

Results

We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS) sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs) and many non crossover (NCO) gene conversions throughout the genome.

Conclusions

By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.