Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes

Abstract

Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information. Phasing short read data typically requires supplemental statistical phasing based on known haplotype structure in the population or parental genotypic data. Here we demonstrate that the MinION nanopore sequencer is capable of producing very long reads to resolve both variants and haplotypes of HLA-A, HLA-B and CYP2D6 genes important in determining patient drug response in sample NA12878 of CEPH/UTAH pedigree 1463, without the need for statistical phasing. Long read data from a single 24-hour nanopore sequencing run was used to reconstruct haplotypes, which were confirmed by HapMap data and statistically phased Complete Genomics and Sequenom genotypes. Our results demonstrate that nanopore sequencing is an emerging standalone technology with potential utility in a clinical environment to aid in medical decision-making.

Phenome-wide association study maps new diseases to the human major histocompatibility complex region

“…It should be noted that other SNP arrays, including Illumina ImmunoChip, 2 provide higher density genotyping for this region compared with the Exome Chip platform. In the future, new long-range sequencing technologies, 49 in combination with the PheWAS approach, may prove necessary to comprehensively study this biologically and clinically important region.…”

Beyond Homopolymer Errors: a Systematic Investigation of Nanopore-based DNA Sequencing Characteristics Using HLA-DQA2

“…Long reads allow for the phased genotyping on polyploid samples, eliminating the need for performing paired-end sequencing with large insert sizes to resolve the gametic phase of distant genetic polymorphisms [13,14].…”

“…This, however was made extremely difficult due to PCR chimera formation, most likely during the second barcoding PCR, reported already in recent papers [13,14]. Partly related to the unique ability of using long strands, PCR chimeras are formed when the complementary strand synthesis is incomplete or when the PCR enzyme complex switches template strands during the amplification.…”

Identifying Polymorphisms in the Alzheimer's Related APP Gene Using the Minion Sequencer

“…The MinION has potential for polymorphism detection when comparing samples; however careful interpretation is needed as high base calling error rates can mask the presence of polymorphisms. to determine cis-haplotypes using the long sequence reads producible by the MinION [5]. Error rates were too high for variant calling using conventional tools such as GATK, therefore polymorphisms were simply identified by classing variant basecalls that occurred in more than a third of total reads as a true SNP.…”

“…Like many of the programme designed to observed polymorphisms within sequences determined by nanopore technology, it aims to control the high basecalling error rate to see whether known polymorphisms could be detected in samples harbouring the variants when aligned to a reference sequence. The tool was unable to detect consensus variants for the deletion and heterozygous samples due to the high background error rate, a factor that has been identified in numerous studies [4,5]. Despite the identification of numerous potential variants including the polymorphisms of interest, against a background of false positives it was not possible to successfully detect variants using this method.…”