2.3 Amplification, sequencing and analysis of MHC genes
The fragment of DRB gene from exon 2 of MHC class II (Beta 1, 231 bp)
was targeted using the primers designed by Becker et al. (2009) forMustela lutreola and a fragment from exon 2 (alpha 1) from MHC
class I, using the primers Meme-MHC-Iex2F and PpLAa1L250 designed by
Sin et al. (2012) for mustelids. PCRs were carried out in 25 μl volumes
containing 0.9 μl of primer mix, 5 μl of GoTaq reaction buffer
(Promega), 2 μl of MgCl2,0.04 μl of BSA, 0.8 μl of
dNTPs, 0.125 μl of GoTaq G2 DNA polymerase (Promega, France) and 3 μl of
DNA. The specific protocol was used for PCR: annealing with touchdown
protocol from 65°C to 56°C for 30 s. Amplified DNA in duplicates were
pooled after quantification using the Quant-iT PicoGreen dsDNA Assay
Kit (Thermo Fisher Scientific Inc., Austria). The library preparation
and sequencing were performed by Novogene (UK). Using their designated
library protocol, 2×250 bp paired‐end sequencing with a depth of 50,000
reads/sample for MHC genotyping and was completed using an Illumina
NovaSeq platform (Illumina Biotechnology Co., Novogene, UK).
To analyze MHC-I and MHC-II amplicon sequences, we used the three-step
pipeline AmpliSAS (Sebastian et al., 2015). Low-quality sequences with
Phred scores lower than 20 were removed and clustering was conducted
using the default parameters for Illumina sequences. Already identified
alleles of MHC-II DRB for E-mink were extracted from NCBI (Becker et
al., 2009), as well as sequences from closely related species
(Mustela putorius and Mustela itatsi ) for MHC-I exon 2. If
NCBI blast reveled 100% of sequence identify between the discovered
alleles in this study and already identified one, their name was
replaced by the accession number of these sequences. For the subsequent
analysis, we focused on the amino acid translated sequences (referred as
MHC motifs) as they are in direct contact with bacteria. We measured
motif richness as the number of sequences per individual for each locus.
We calculated functional distances between individuals following the
approach described in Strandh et al. (2012). A maximum-likelihood tree
was constructed based on the chemical binding properties of the amino
acids, as described by five physico-chemical descriptor variables
(z-descriptors) for each amino acid, using sequences of Meles
meles , Meles leucurus , Meles anakuma and Martes
zibelina as out-group retrieved through NCBI blast (Figure S1). The
trees were used as reference from which the functional distances between
individuals were calculated using unweighted UniFrac for both genes
(Lozupone & Knight, 2005). Following Bolnick et al. (2014), the genetic
distance between each amino acid sequences within each individual
(Faith’s PD) were calculated, and further defined as motif divergence.