Kathrin Lang

ABO allele-level frequency estimation based on population-scale genotyping by next generation sequencing

BackgroundThe characterization of the ABO blood group status is vital for blood transfusion and solid organ transplantation. Several methods for the molecular characterization of the ABO gene, which encodes the alleles that give rise to the different ABO blood groups, have been described. However, the application of those methods has so far been restricted to selected samples and not been applied to population-scale analysis.ResultsWe describe a cost-effective method for high-throughput genotyping of the ABO system by next generation sequencing. Sample specific barcodes and sequencing adaptors are introduced during PCR, rendering the products suitable for direct sequencing on Illumina MiSeq or HiSeq instruments. Complete sequence coverage of exons 6 and 7 enables molecular discrimination of the ABO subgroups and many alleles. The workflow was applied to ABO genotype more than a million samples. We report the allele group frequencies calculated on a subset of more than 110,000 sampled individuals of German origin. Further we discuss the potential of the workflow for high resolution genotyping taking the observed allele group frequencies into account. Finally, sequence analysis revealed 287 distinct so far not described alleles of which the most abundant one was identified in 174 samples.ConclusionsThe described workflow delivers high resolution ABO genotyping at low cost enabling population-scale molecular ABO characterization.

Accurate typing of class I human leukocyte antigen by Oxford nanopore sequencing

Oxford Nanopore Technologies’ MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunological compatibility between organ donors and recipients, but the platform’s high error rate makes it challenging to type alleles with clinical-grade accuracy. Here, we developed and validated Athlon, an algorithm that iteratively scores nanopore reads mapped to a hierarchical database of HLA alleles to arrive at a consensus diploid genotype; Athlon achieved a 100% accuracy in class I HLA typing at high resolution.

OR38 Whole gene HLA class I genotyping with the minion nanopore sequencer

In 2014 Oxford Nanopores Technologies released the MinION, a portable miniaturized sequencer delivering multi-kb DNA reads based on nanopore sensing technology. Sequencing accuracy is improved by consecutive sequencing both strands of the DNA (2D reads). Being part of the MinION early access program we evaluated this new technology for whole gene HLA sequencing. As the sequencing yield exceeds the requirements for a targeted approach we developed a strategy for barcoding and demultiplexing of samples. Barcoded amplicons were generated by two subsequent long range PCRs covering the complete genes of HLA-A, -B and -C. Amplicons were pooled and library preparation was performed according to the Oxford Nanopore protocol (SQK-MAP006). Sequencing was performed on a MinION MKI with R7.3 flow cells. NGSengine from GenDX was used for sequence analysis and HLA genotyping. 96 randomly picked class I samples were pooled and sequenced. Over 170,000 reads were generated, approximately 65% of them as 2D reads. Two thirds of the 2D reads could be assigned to a unique barcode sequence. The HLA genotyping results were compared to allelic resolution genotypes obtained by full length sequencing the samples on both Illumina and PacBio instruments. The ONT typing results had a 95% concordance rate when considering 1st to 3rd field. Even when taking the fourth field in account concordance rate was above 90%. Given that the nanopore technology is rapidly evolving and the genotyping software has not yet been optimized for nanopore data, this study clearly underlines the great potential of the MinION for high resolution HLA genotyping.