This is a repository to accompany the manuscript "Genetic and epigenetic insight into morphospecies in a reef coral".

The study involves analysis of ddRAD-seq and EpiRAD-seq data for 27 samples of branching Porites spp. corals collected in Belize. There is taxonomic uncertainty whether Caribbean branching Porites spp. comprise 3 different species or a single polymorphic species. The species are distinguished primarily by branch thickness.

The goal is to evaluate (1) if there is any genetic structuring of these individuals using the ddRAD data and (2) if there is any epigenetic structuring of these individuals using the EpiRAD data.

A starting set of 48 samples of Porites spp. were prepared for ddRADseq and EpiRADseq. Only 30 of these samples were the branching Porites spp. analyzed in this study; the rest were Porites astreoides destined for analysis in a separate study. A separate ddRAD and EpiRAD library was created for each sample, meaning that a total of 96 (48 ddRAD and 48 EpiRAD) samples were run on a 96 well plate. These were split into 12 pools that were run on a single Illumina HiSeq 4000 lane (100 bp paired-end reads). The data directory contains sample metadata and barcode files associated with each library.

Sequences were assembled using ipyrad v0.3.41 (Eaton 2014). We used the ‘denovo - reference’ assembly method with the Symbiodinium minutum (clade B; (Shoguchi et al. 2013)) and Symbiodinium kawagutii (clade F; (Lin et al. 2015) ) draft genomes used as reference to subtract symbiont reads from the de novo assembly. The assembly workflow details the assembly in a Jupyter notebook. Further specifics of the ipyrad parameters used for assembly can be seen in the aforementioned workflow or directly in the parameters file. Information on these parameters and how they are used in ipyrad can be found here.

Each sample has both a ddRAD-seq and EpiRAD-seq library associated with it. The only difference between the libraries is that the ddRAD library was generated with a methylation-insensitive common cutter (MspI) while the EpiRAD library was generated with a methylation-sensitive common cutter (HpaII). Both restriction enzymes recognize the same cut site, CCGG, but if this site is methylated, HpaII will not cut and the locus will not be present in the EpiRAD library. Both libraries had the same rare cutter (PstI). Thus, both libraries should contain similar sets of loci unless the locus is methylated.

In essence the analysis involves (1) analysis of single nucleotide polymorphisms (SNPs) in the ddRAD data and (2) analysis of read counts in the EpiRAD data. ipyrad generates many output files that can be readily used for the ddRAD SNP anlysis, but the EpiRAD analysis requires a bit more work to extract workable data. First, read count data are extracted from the .vcf format output from iPyrad. This file is too large to be included in the online repository, but the workflow can be found here. The remainder of the analysis is performed in R. The R notebook is located here.

This study made use of the following software:

ipyrad v0.3.41

SnpEff(SnpSift) v4.2

R v3.1.3

RStudio v1.0.136

ape v3.4

edgeR v3.8.6

adegenet v2.0.1

ade4 v1.7-4

hierfstat v0.04-22

lmtest v0.9-34

relaimpo v2.2-2

iheatmapr v0.2.5

reshape2 v1.4.2

ggplot2 v2.2.1

analyses/ - Files resulting from ipyrad assembly and R analyses.

data/ - Metadata and links to methods and raw data.

images/ - Coral photos, gel images, figures.

notebooks/ - Jupyter notebooks.

scripts/ - Scripts such as R scripts used for analyses.