12-OR: IN VITRO GENERATED PCR CROSSOVER PRODUCTS IDENTIFIED BY NEXT GENERATION SEQUENCING CORRESPOND TO SOME ALLELES IN THE IMGT DATABASE

摘要: Aim Genotyping of DRB1 and DRB3/4/5 is performed in our lab using generic DRB PCR primers, followed by sequencing on the 454 GS FLX use Conexio Genomics software. During genotyping homozygous cell lines, we detected additional sequences at about 7% read depth reported true allele. These artifacts appeared to be crossovers between sequences. The usually had one or more mismatches with IMGT database but occasionally matched a very rare allele (e.g. ∗ 03:42). We study determine if these were vitro some alleles might, fact, that been submitted. Methods To test for artifact formation during late cycles genomic PCR, amplified loci from 21 cells lines under both standard conditions (35 cycles) reduced number (28), sequencing. Using same conditions, also sequenced four samples, each which previously identified as having novel was subsequently submitted database. Results For all amplification 35 gave low abundance possible “second alleles.” In 36% cases, artifactual corresponded named alleles. Such not 28 cycles. case 4 samples IMGT, found 3 fourth, while crossover product, contained error revealed HLA assay. Conclusions Formation products can occur PCR. have minimize generation artifacts. Clonal valuable method revealing traditional errors.