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

ABSTRACT: 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.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.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.

SUBMITTER: Samarakoon U

PROVIDER: S-EPMC3055840 | biostudies-other | 2011 Feb

REPOSITORIES: biostudies-other

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

Samarakoon Upeka U Regier Allison A Tan Asako A Desany Brian A BA Collins Brendan B Tan John C JC Emrich Scott J SJ Ferdig Michael T MT

BMC genomics 20110217

<h4>Background</h4>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.<h ...[more]

PMID: 21324207

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Project description:We used a high-density tiling array to estimate genetic recombination rate among 32 independent recombinant progeny of a P. falciparum genetic cross (7G8 M-CM-^W GB4). We detected 3184 segregating multi-probe single-feature polymorphisms (mSFPs) and 638 recombination events (496 excluding those from subtelomeric regions). These data, in combination with results from 254 previously reported microsatellites, enabled us to construct a high-resolution genetic map. Comparing genetic and physical maps, we obtained an overall recombination rate of 9.6 kb/cM (12.8 kb/cM excluding subtelomeric regions) and identified 54 hotspots, some of which occurred in genes encoding surface antigens or proteins with repetitive motifs that might play a role in genetic recombination in the parasite. Motifs enriched in hotspots were also identified. In agreement with results from a previous cross (HB3 M-BM-4 Dd2), there was positive correlation between sizes of individual chromosomes and their recombination events. These results show that the P. falciparum genome is highly recombinogenic, providing an important genetic basis for parasite survival under various selection pressures. GC-rich repetitive motifs identified in the hotspot sequences may play a role in the high recombination frequency observed. Ten microgram of genomic DNA, extracted and purified from 3D7 (reference), thirty-two P. falciparum independent recombinant progeny of the 7G8 x GB4 cross, and the two parental lines (Hayton, 2008), were hybridized to the PFSANGER GenechipM-BM-. (Affymetrix, Inc., Santa Clara, CA, USA). The scanned image CEL files were first processed using the RMA method, then averaged and compared with reference genome 3D7, and lastly assigned either 7G8 or GB4 alleles based on similarities to the two parental lines. Total of 35 genomic DNA samples (biological replicates: 6 for 3D7, 4 for 7G8, 4 for GB4, and 2 for Pf_WE2). The supplementary file 'GSE25656_QuantNormData_Log2_AllSamples.txt' contains the RMA-normalized data for all of the samples. The supplementary files 'GSE25656_chr*' contain the parental allele assignment of each chromosome and include probe-level annotation.

Dataset Information

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

Publications

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

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