DOI: 10.5061/dryad.qfttdz0qb
title: Template-specific optimization of NGS genotyping pipelines reveals allele-specific variation in MHC gene expression

description: Using high-throughput sequencing for precise genotyping of multi-locus
 gene families, such as the Major Histocompatibility Complex (MHC), remains
 challenging, due to the complexity of the data and difficulties in
 distinguishing genuine from erroneous variants. Several dedicated
 genotyping pipelines for data from high-throughput sequencing, such as
 next-generation sequencing (NGS), have been developed to tackle the
 ensuing risk of artificially inflated diversity. Here, we thoroughly
 assess three such multi-locus genotyping pipelines for NGS data, the DOC
 method, AmpliSAS and ACACIA, using MHC class IIβ datasets of three-spined
 stickleback gDNA, cDNA, and “artificial” plasmid samples with known
 allelic diversity. We show that genotyping of gDNA and plasmid samples at
 optimal pipeline parameters was highly accurate and reproducible across
 methods. However, for cDNA data, gDNA-optimal parameter configuration
 yielded decreased overall genotyping precision and consistency between
 pipelines. Further adjustments of key clustering parameters were required
 tο account for higher error rates and larger variation in sequencing depth
 per allele, highlighting the importance of template-specific pipeline
 optimization for reliable genotyping of multi-locus gene families. Through
 accurate paired gDNA-cDNA typing and MHC-II haplotype inference, we show
 that MHC-II allele-specific expression levels correlate negatively with
 allele number across haplotypes. Lastly, sibship-assisted cDNA-typing of
 MHC-I revealed novel variants linked in haplotype blocks and a
 higher-than-previously-reported individual MHC-I allelic diversity. In
 conclusion, we provide novel genotyping protocols for the three-spined
 stickleback MHC-I and -II genes and evaluate the performance of popular
 NGS-genotyping pipelines. We also show that fine-tuned genotyping of
 paired gDNA-cDNA samples facilitates amplification bias-corrected MHC
 allele expression analysis.
description: # Template-specific optimization of NGS genotyping pipelines reveals
 allele-specific variation in MHC gene expression
 ## Description of the data
 and file structure This submission consists of two Excel files. The file
 'Data_MHC-I' includes information regarding the 10 three-spined
 stickleback families included in our MHC-I genotyping dataset, and is
 separated into three sheets: (i) Families overview, with information
 regarding the number of offspring and individual IDs of the families
 (columns: family ID, and corresponding offspring IDs) (ii) Family
 genotypes (columns: Family ID, Inferred Parental Genotype1, Inferred
 Parental Genotype2, Observed Offspring Genotypes, Number of Alleles Per
 Genotype, and Number of Offspring), and (iii) Allele segregation by
 family, where a table is presented for each of the 10 families used to
 infer the genetic linkage between MHC-I loci of the three-spined
 stickleback. The file 'Data_MHC-II' includes the genotypes of
 all samples included in our MHC-II genotyping dataset. It is structured in
 one sheet (columns: Sample ID, Sample template, Data subset, Genotype, and
 Depth, followed by the names and corresponding depth (in reads) of all
 alleles called in each sample. All genotypes described were obtained at
 optimal AmpliSAS configuration for each template type (gDNA, cDNA,
 plasmid), as described in detail in our paper.