Template-specific optimization of NGS genotyping pipelines reveals allele-specific variation in MHC gene expression

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. F..., , , # 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 M...