Authors:
B. Matern (Maastricht, NL, NL)
M. Groeneweg (Maastricht, NL)
T. Slangen (Maastricht, NL)
C. Meertens (Maastricht, NL)
C. Voorter (Maastricht, NL)
M. Tilanus (Maastricht, NL)
In the years past, MinION has often been dismissed as a useful platform for clinical diagnostics, citing high per-base error rates and lack of analysis tools as the main apprehensions. Recently, the technology has advanced rapidly, and we regularly observe reads with an average Phred score of Q=20, corresponding to 99.0% per-base accuracy. Oxford Nanopore releases regular updates on the sequencing and basecalling platforms, and members of the MinION community continually develop improvements on analysis and assembly tools. De novo assembly of nanopore reads remains a challenge, but data analysis has reached a point where reads are useable in analysis of complicated polymorphic regions, such as HLA. Our goal is to develop a high-throughput, highly accurate open-source analysis tool for HLA allele calling based on initial de novo assembly, followed by alignment to sequences from the IPD-IMGT/HLA Database. This analysis pipeline uses both community and in-house software in a structured way to sort, assemble, and classify high-quality Nanopore reads for applications in research, and ultimately routine diagnostics. As a proof of concept, we sequenced the full-length classical class I HLA genes (A,B,C) of three individuals with potentially novel HLA types. The MinION reads were analyzed, filtered for quality and length, and assembled into consensus sequences for each of the three class I loci. The sequences were aligned to allele references available in the IPD-IMGT/HLA Database for comparison with known alleles. For automated submission of novel full-length HLA alleles to the EMBL-ENA Database, an in-house allele submission tool was generated, and is available for general public use. Overall, the developed analysis tool represents a promising method for analysis of MinION data and assignment of HLA alleles based on full-gene Nanopore sequences.