Genome sequencing and bioinformatics
DNA was extracted from blood, using Qiagen MagAttract, linked read library preparation was 10x Genomics Chromium technology, which were then sequenced on an Illumina HiSeq X with 2x150bp reads . The sequencing read data was mapped to a previously generated pink pigeon reference genome . The variant calls were used to create a per-SNP pink pigeon CADD (ppCADD) score calculated for the UCEs of each individual’s genome (Figure 1). A Snakemake pipeline allowing for reproduction of this approach can be found on GitHub (https://github.com/saspeak/LoadLift).
Previously published tetrapod ultraconserved element (UCE) probes based on the chicken reference genome and the Tibetan ground-jay (Pseudopodoces humilis ) were used to harvest UCEs from the pink pigeon reference genome, using the Phyluce workflow . A chain file was created for annotation lift-over and the CADD scores of the chicken genome were cross mapped to the reference pigeon genome using CrossMap.py . CADD scores were filtered to remove non-scoring and fixed sites. Genotypes of each locus were assessed to calculate the genetic load components. Individual’s genetic load, realized load and masked load were calculated using the following formulas :
\begin{equation} Genetic\ load\ (individual\ k)=\sum_{i=1}^{L(hom)}s_{i}+\sum_{j=1}^{L(het)}{0.5}s_{j}\nonumber \\ \end{equation}
[1]
\begin{equation} \text{Realised~{}load~{}}\left(\text{individual\ k}\right)=\sum_{i=1}^{L(hom)}s_{i}+\sum_{j=1}^{L(het)}{h_{j}s_{j}}\nonumber \\ \end{equation}
[2]
\begin{equation} Masked\ load\ (individual\ k)=\sum_{j=1}^{L(het)}{(0.5-}{h_{j})s}_{j}\nonumber \\ \end{equation}
[3]
Here, si (and sj ) is the ppCADD score at locus i (and j ), and they are summed across all homozygous (or heterozygous) loci at the UCEs of individual k. In the computer simulations (see below), s and h stand for the selection and dominance coefficients, and the fitness impact of the load can be expressed in lethal equivalents . For simplicity, the dominance coefficient (hj ) is assumed to behj =0.1. Noted that part of the realised load comprises heterozygous mutations that are assumed to be partially dominant. Inbreeding coefficients (FRoH) of the six pink pigeons were calculated using runs of homozygosity (RoH) with bcftools roh . For further details, see Supplementary Information.