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.