title: Identification of piggyBac-mediated insertions in Plasmodium berghei by next generation sequencing.
abstract: BACKGROUND
  The piggyBac transposon system provides a powerful forward genetics tool to study gene function in Plasmodium parasites via random insertion mutagenesis and phenotypic screening. The identification of genotype of piggyBac mutants in the Plasmodium genome is thus an indispensable step in forward genetic analysis. Several PCR-based approaches have been used to identify the piggyBac insertion sites in Plasmodium falciparum and Plasmodium berghei, but all are tedious and inefficient. Next generation sequencing can produce large amounts of sequence data and is particularly suitable for genome-wide association studies. In this study, the Next generation sequencing technology was employed to efficiently identify piggyBac insertion sites in the genome of P. berghei.
  
  
  METHODS
  Plasmodium berghei parasites were co-transfected with piggyBac donor and helper plasmids. Initially, the classical inverse PCR method was used to identify the existence of piggyBac insertions in the P. berghei genome. The whole genome of post-transfection parasites was subsequently sequenced with a PCR-free paired-end module using the Illumina HiSeq sequencing system. The two distinct methods ('BLAST method' and 'SOAP method') were employed to identify piggyBac insertion sites in the P. berghei genome with Illumina sequencing data. All the identified piggyBac insertions were further tested by half-nested PCR.
  
  
  RESULTS
  The inverse PCR method resulted in a very low yield of ten individual insertions identified. Conversely, 47 piggyBac insertions were identified from about 1 Gb of Illumina sequencing data via the two distinct analysis methods. The majority of identified piggyBac insertions were confirmed by half-nested PCR. In addition, 1,850 single nucleotide polymorphisms were identified through alignment of the Illumina sequencing data of the P. berghei ANKA strain used in this study with the reference genome sequences.
  
  
  CONCLUSIONS
  This study demonstrates that a high-throughput genome sequencing approach is an efficient tool for the identification of piggyBac-mediated insertions in Plasmodium parasites.
authors: Department of Tropical Infectious Diseases, Second Military Medical University, Shanghai, China.
authors: Yi
authors: Y
authors: Cao
authors: Bing
authors: B
authors: Rui
authors: Dianne L
authors: DL
authors: Wellems
authors: Mingxing
authors: M
authors: Li
authors: Biaobang
authors: B
authors: Chen
authors: Dongmei
authors: D
authors: Zhang
authors: Weiqing
authors: W
authors: Pan
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D018503:Genome, Protozoan; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D051379:Mice; D008967:Molecular Biology / Q000379:methods; D016254:Mutagenesis, Insertional; D010957:Plasmids; D010962:Plasmodium berghei / Q000235:genetics*; D011995:Recombination, Genetic
chemical_list: D004251:DNA Transposable Elements; D016054:DNA, Protozoan
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