ASSIGN: A COMPLETE SOFTWARE PACKAGE FOR ALLELE ASSIGNMENT AND QUALITY CONTROL OF DNA SEQUENCING BASED TYPING

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TITLE: ASSIGN: A COMPLETE SOFTWARE PACKAGE FOR ALLELE ASSIGNMENT AND QUALITY CONTROL OF DNA SEQUENCING BASED TYPING

David C. Sayer,^1,2,3 Damian M. Goodridge.^1,3

¹Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Australia; ²Pathology, University of Western Australia, Western Australia, Australia; ³Connexia Genomics, Perth, Western Australia, Australia

DNA sequence analysis and the deduction of HLA alleles from DNA sequence remains a bottleneck for high throughput DNA sequencing based HLA typing (SBT). We have developed a software package called Assign that removes this bottleneck. The primary function of Assign is to deduce the HLA alleles from DNA sequence. However Assign contains additional features critical for the quality control of SBT. Sequence text files or files containing Phred quality values (PQV) can be analysed. Analysis of sequence with PQV is a significant advance for SBT. QC analysis of entire sequence runs, sequences from specific samples or individual sequence reactions can be performed. PQV fall into three categories. Correct base calls at sites containing a single nucleotide, correct heterozygous base calls and heterozygous sequence which has been incorrectly called as containing a single nucleotide. Only positions within a sequence with a PQV indicating an incorrect base call require manual review. This reduces editing time considerably and only a few (frequently none) sites within a sequence require review. An additional QC feature of Assign is the detection of preferential amplification of one allele over another. This is critical for the evaluation of new reagents, reagent batches, protocols and the detection of those alleles with primer site polymorphisms. Allele assignment is performed following comparison of the test sequence with a library generated from sequences from the IMGT database. Results are generated in an easily interpreted format that lists the location of sequence differences between the best matched and the test sequence. Additional features of Assign include reporting serological specificities and the identification of primer sites for resolution of ambiguities by SBT. This feature combines the primer information and the sequence to produce an unambiguous allele report. Assign combines quality control features with allele assignment for all HLA loci. Assign eliminates sequence analysis and editing as a bottleneck for SBT.