Application of high-throughput next-generation sequencing for HLA typing of DNA extracted from postprocessing cord blood units.

Cord blood has become an acceptable source of hematopoietic stem cells for transplantation. HLA plays a major role in hematopoietic stem cell transplantation (HSCT). Typing of cord blood samples for HLA alleles has been performed based on the serological and molecular methods. However, with the advent of next-generation sequencing technology, HLA typing becomes more accurate and unambiguous (upto intron level). Contamination of cord blood cells with erythropoietic cells poses a challenge in DNA extraction and downstream application. In the present study, DNA extracted from buffy coat of cord blood samples was typed for HLA-A, -B, -C, DRB1, and DQB1 alleles by Illumina miniseq and the sequences were aligned, phased, and mapped by MIA FORA software algorithms. Most frequent alleles found were HLA A*01:01:01 (17%), A*24:02:01 (15.1%), A*11:01:01 (13.6%), B*40:06:01 (10.7%), C*06:02:01 (17.7%), C*04:01:01 (14.2%), C*15:02:01 (11.4%), C*07:02:01 (10.7%), DRB1*07:01:01 (15.9%), DRB1*10:01:01 (10.2%), DQB1*06:01:01 (17.4%), DQB1*05:01:01 (12.4%), and DQB1*05:03:01 (10.4%). One null allele (A*24:11N), two novel alleles in B loci and three rare alleles (B*40:06:04, B*51:01:05, and C*01:44) were also identified in the present study. This study shows that high-throughput, unambiguous (third-field resolution) HLA typing can be performed on cord blood samples. In order to preserve the precious sample for future use, minimal amount of cord blood samples (postprocessing) could be used for HLA typing purpose.

Identification of the novel HLA allele, HLA-B*35:03:23, by next-generation sequencing.

The novel HLA-B allele, HLA-B*35:03:23, differs from B*35:03:01 by a single-nucleotide mismatch in exon 1.