MHC class II DRB exon 2 gene from Murinae (Rodentia). Rodentia

High-throughput sequencing technologies offer new perspectives for biomedical, agronomical andevolutionary research. Promising progresses now concern the application of these technologies to large-scale studiesof genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possibleusing 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise:first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguishtrue from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed todemonstrate the reliability of this method. DNA amplicons from 1,575 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 171 bp fragments (excluding primers and tags) corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 364,442 reads were obtained, of which 249,186 were finally assigned to original samples using SESAME software.