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Rapid, highly accurate and cost‐effective open‐source simultaneous complete HLA typing & phasing of Class I & II alleles using nanopore sequencing

Publication

Date: 18th May 2020 | Source: HLA

Authors: Joanne D. Stockton, Thomas Nieto, Elizabeth Wroe, Anthony Poles, Nicholas Inston, David Briggs, Andrew D. Beggs.

Introduction
Accurate rapid genotyping of the genes within the HLA region presents many difficulties due to the complexity of this region.

Here we present the results of our proof of concept Nanopore-based long read PCR solution for HLA genotyping.

Methods
For 15 HLA Anthropology based samples and 13 NHS Blood and Transplant derived samples 40 ng of genomic DNA underwent long range PCR for class I and II HLA alleles. Pooled PCR products were sequenced on the Oxford Nanopore MinION R9.4.1 flow cell. Sequenced reads had HLA genotype assigned with HLA‐LA. Called genotypes were compared to reference derived from a combination of short read NGS, Sanger sequence and/or SSP typing.

Results
For concordance, accuracy was 100%, 98.4%, 97.5% and 95.1% for the first, second, third and fourth fields respectively to 4 field accuracy where it was available, otherwise 3 field) in 28 samples for class I calls and 17 samples for class II calls.

Phasing of maternal and paternal alleles, as well as phasing based identification of runs of homozygosity was demonstrated successfully.

Time for assay run was 8 hours and reconstruction of HLA typing data was 15 minutes. Assay cost was £55 ($80USD)/sample.

Conclusions
We have developed a rapid and cost‐effective long range PCR and nanopore sequencing based assay that can genotype the genes within HLA region to up to 4 field accuracy, identify runs of homozygosity in HLA, reconstruct maternal and paternal haplotypes and can be scaled from multi‐sample runs to a single sample.

Read the full text Watch Tom Nieto's NCM18 talk on rapid HLA typing

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