title: Whole genome profiling of spontaneous and chemically induced mutations in Toxoplasma gondii.
abstract: BACKGROUND
  Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies. One traditionally intractable group, the Apicomplexa, contains several important pathogenic protozoan parasites, including the Plasmodium species that cause malaria.Here we apply whole genome analysis methods to the relatively accessible model apicomplexan, Toxoplasma gondii, to optimize forward genetic methods for chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and ethylmethane sulfonate (EMS) at varying dosages.
  
  
  RESULTS
  By comparing three different lab-strains we show that spontaneously generated mutations reflect genome composition, without nucleotide bias. However, the single nucleotide variations (SNVs) are not distributed randomly over the genome; most of these mutations reside either in non-coding sequence or are silent with respect to protein coding. This is in contrast to the random genomic distribution of mutations induced by chemical mutagenesis. Additionally, we report a genome wide transition vs transversion ratio (ti/tv) of 0.91 for spontaneous mutations in Toxoplasma, with a slightly higher rate of 1.20 and 1.06 for variants induced by ENU and EMS respectively. We also show that in the Toxoplasma system, surprisingly, both ENU and EMS have a proclivity for inducing mutations at A/T base pairs (78.6% and 69.6%, respectively).
  
  
  CONCLUSIONS
  The number of SNVs between related laboratory strains is relatively low and managed by purifying selection away from changes to amino acid sequence. From an experimental mutagenesis point of view, both ENU (24.7%) and EMS (29.1%) are more likely to generate variation within exons than would naturally accumulate over time in culture (19.1%), demonstrating the utility of these approaches for yielding proportionally greater changes to the amino acid sequence. These results will not only direct the methods of future chemical mutagenesis in Toxoplasma, but also aid in designing forward genetic approaches in less accessible pathogenic protozoa as well.
authors.forename: Andrew
authors.initials: A
authors.lastname: Farrell
authors.forename: Bradley I
authors.initials: BI
authors.lastname: Coleman
authors.forename: Brian
authors.initials: B
authors.lastname: Benenati
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Brown
authors.forename: Ira J
authors.initials: IJ
authors.lastname: Blader
authors.forename: Gabor T
authors.initials: GT
authors.lastname: Marth
authors.affiliation: Department of Biology, Boston College, Higgins Hall 355, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA. gubbelsj@bc.edu.
authors.forename: Marc-Jan
authors.initials: MJ
authors.lastname: Gubbels
mesh_terms: D000241:Adenosine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D020029:Base Pairing; D002460:Cell Line; D005020:Ethyl Methanesulfonate / Q000633:toxicity; D005038:Ethylnitrosourea / Q000633:toxicity; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000235:genetics / Q000378:metabolism; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics*
chemical_list: D015800:Protozoan Proteins; D005020:Ethyl Methanesulfonate; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase; D000241:Adenosine; D005038:Ethylnitrosourea; D013936:Thymidine