P066 Next-generation sequencing (NGS) HLA typing: Beyond allele assignment

Abstract

Aim To expand the analytical potential of NGS in HLA typing. Studies based on SNP analysis have revealed the DNA sequence of HLA genes to carry important biological information typically overlooked in the simple exercise of allele assignment. NGS software is optimised to match sample sequences against databases of HLA allele sequence references. The goal here, however, is to create a method to open the data collected by NGS to any kind of query. Methods BLAST is still one of the most robust and efficient sequence-matching and sequence-alignment methods. Typically BAST databases are built with reference sequences against which a sample sequence is queried for matches. Here we reverse the approach and build a database of sample sequences against which we query for matches for particular sequences of interest following these steps: 1. Compile a database of sample sequences included in a given study. 2. Convert sample sequences to FASTA format. 3. Create a BLAST database with the study sample sequences. 4. Run any BLAST query against the BLAST database of the study sample sequences. Results This method was implemented using a dataset collected for typing purposes. The HLA-C 3’UTR polymorphisms which bind to miRNAs and regulate the level of expression (Nature 2011; 472:7344) were used. The BLAST output provided accurate information about which sequences carried the query polymorphism, which matched what is known about the association of these SNPs with HLA-C alleles. This method has also been successfully used to look for mutations that render HLA alleles null, and other SNP analysis queries. Conclusions NGS provides data that goes beyond the need for simple allele assignment. The method presented here provides (1) a robust and reliable way to store this accumulated information, (2) a quick and simple way to query this database of sequence data; and (3) an open method to ask any sequence question. M. Li: Employee; Company/Organization; Immucor.