The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci is challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II-DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)

S. Fuselli
Primo
;
A. Panziera;A. Benazzo;G. Bertorelle
Ultimo
2018

Abstract

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci is challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II-DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.
Fuselli, S.; Baptista, R. P.; Panziera, A.; Magi, A.; Guglielmi, S.; Tonin, R.; Benazzo, A.; Bauzer, L. G.; Mazzoni, C. J.; Bertorelle, G.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2385324
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