Testing genotyping strategies for ultra‐deep sequencing of a co‐amplifying gene family: MHC class I in a passerine bird

Abstract

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.

Lack of statistical power as a major limitation in understanding MHC‐mediated immunocompetence in wild vertebrate populations

“…Compared to other gene systems, the combination of the highly dynamic evolution of the MHC system (occurrence of many duplicated genes, high rates of recombination and gene conversion, and high levels of polymorphism) and errors generated during PCR and sequencing make it difficult to discriminate artefacts from true MHC alleles (Babik, ; Burri, Promerova, Goebel, & Fumagalli, ). However, the advent of high‐throughput sequencing technology coupled with recent filtering procedures specifically designed for the MHC system have considerably helped to gain reliable genotypes (Babik, Taberlet, Ejsmond, & Radwan, ; Biedrzycka, Sebastian, Migalska, Westerdahl, & Radwan, ; Ferrandiz‐Rovira, Bigot, Allaine, Callait‐Cardinal, & Cohas, ; Gaigher et al, ; Grogan, McGinnis, Sauther, Cuozzo, & Drea, ; Lighten et al, ; Oomen, Gillett, & Kyle, ; Promerová et al, ; Rekdal, Anmarkrud, Johnsen, & Lifjeld, ; Sebastian, Herdegen, Migalska, & Radwan, ; Sommer et al, ; Stutz & Bolnick, ). Such technology also enables the sequencing of thousands of samples in parallel, which can drastically improve sample sizes for association studies.…”

“…Such technology also enables the sequencing of thousands of samples in parallel, which can drastically improve sample sizes for association studies. To date, Illumina MiSeq technology is the method of choice and has proven to bring a great value for MHC genotyping due to its high sequencing depth (Gaigher et al, ; Herdegen, Babik, & Radwan, ; Lighten et al, ), even in species with a large number of co‐amplified alleles (Biedrzycka et al, ). Finally, coupled with high sequencing coverage, family‐based studies can significantly contribute to accurate MHC genotyping (Gaigher et al, ).…”

Blood parasites shape extreme major histocompatibility complex diversity in a migratory passerine

“…Ultra‐deep sequencing technologies that become recently available are essential for working with such highly complex gene families (Biedrzycka, Sebastian et al., ). We used amplicon sequencing with Illumina Miseq technology to assess the diversity of MHC class I exon 3 in sedge warbler.…”

“…Processing of Illumina data was based on the earlier work of Biedrzycka, Sebastian et al. (), who found that among four tested methods, the adjustable clustering method (Sebastian, Herdegen, Migalska, & Radwan, ) gave the most accurate genotyping results. All amplicon sequencing analysis tools used are available at http://evobiolab.biol.amu.edu.pl/amplisat/.…”

“…The sedge warbler, Acrocephalus schoenobaenus , is a passerine bird species that has more than 30 MHC class I gene copies, a gene copy number that is much higher than in studies that have found an optimal number of MHC genes (Kloch et al., ; Wegner, Kalbe, Kurtz, Reusch, & Milinski, ). Moreover, the number of MHC class I alleles per bird varies considerably between individuals (mean = 36.3, range = 12–65, Biedrzycka, Sebastian et al., ). Such a broad range cannot be explained solely by zygosity differences between individuals and must at least partly be due to copy number variation.…”