**In this repository following Supplementary Datasets accompanying Article: “Evolutionary trade-offs constraining the MHC gene expansion: beyond simple TCR depletion model” by Migalska et al. 2024, Front. Immunol. have been deposited:**
1. MHC_demultiplexing_files.zip
Files contain primer combinations and 6-bp long tags necessary for de-multiplexing of pooled MHC amplicon libraries stored at PRJNA1003007 (BioSamples: SAMN36871034-5, SRA: SRX21277855, SRX21277825). Samples can be demultiplexed with e.g., AmpliSAS software.
2. SI_Intermediate_genotyping_files.xlsx
Juxtaposition of provisional genotypes (with read counts) obtained with AmpliSAS (after de-multiplexing, clustering and filtering of reads) for each of the four MHC markers (i.e., regions amplified by a primer pair: MHC class I exon 3, MHC class II exon 2 of DQA, DQB and DRB), in four PCR amplicons: R1A, R1B, R2A, R2B. For each individual template for PCR reactions was cDNA from one of two independent RNA extractions: R1 and R2. For each individual, R1 (SRX21277825) was an extract from spleen cells (i.e., aliquot #3 in the Animals and tissue processing section in the Methods section of the article) or from a liver fragment (when the amount of splenocytes did not suffice for a successful RNA extraction); R2 (SRX21277855) was an extract from a liver fragment (another liver fragment, if R1 was also liver-based). Subsequently, each of the four markers was amplified in four PCR reactions – two using R1 material as a template (i.e., R1A, R1B), and two using R2 as a template (i.e., R2A, R2B).
For final genotyping (reported in the Supplementary Dataset 1, alongside published article – doi: 10.3389/fimmu.2023.1240723), variants were called for each marker when present in at least two out of four amplicons (basic “two-in-four” genotyping criterion) with more than 3 reads in each. Additional genotyping criteria were taken into account for MHC class I (explained in Supplementary Material file associated with the article and also below).
3. SI_Features_MHCI.xlsx:
MHC class I genotyping involved additional steps to exclude potential non-classical MHC class I genes (see Supplementary Materials in Supplementary Information: Materials, Tables and Figures file at https://www.frontiersin.org/articles/10.3389/fimmu.2023.1240723/abstract#supplementary-material for further details). This file provides information on certain features associated with detected, nucleotide MHC class I variants, i.e.,: I) anchoring residues (column “anchoring”) from amino acid translation of the sequence and II) tissue expression pattern (column “tissue”) that was used to refine final MHC class I genotypes.
I) Presence of certain conserved, peptide-anchoring residues was proposed to characterize classical, MHC Ia genes [Kaufman et al. 1994, Semin Immunol 6:411–424]. Our MHC class I amplicons covered five (out of nine proposed) such residues: T143, K146, W147, Y159 and Y171. Column “anchoring” summarizes amino acids at these positions for each sequence (i.e., “canonical pattern is ”TKWYY”).
II) Tissue-specific expression could cause systematic differences in gene number estimation between individuals for which only liver was used in MHC genotyping, versus those that used two tissue types (i.e., liver and spleen). To avoid such bias, we excluded variants that consistently were amplified only from liver (“lv”) or spleen (“sp”), and never from both tissues (“both”).
