This repository contains all of the raw data, code, and supplemental information for the manuscript:

Using high-throughput sequencing of ITS2 to describe Symbiodinium metacommunities in St. John, U.S. Virgin Islands

Authors: Ross Cunning, Ruth D. Gates, Peter J. Edmunds

Journal: PeerJ

Link:

---

Description:

This project investigates Symbiodinium communities in scleractinian and milleporine corals at St. John, US Virgin Islands using high-throughput sequencing of Symbiodinium ITS2 nrDNA. The major aims of this work are to:

1. Apply a novel bioinformatic approach (within-sample clustering) to better understand Symbiodinium ecology using ITS2 metabarcoding data

2. Characterize Symbiodinium communities in a range of coral species using high-throughput sequencing (many for the first time)

The bioinformatic pipeline* is coded using a Makefile that calls a series of Shell and R scripts that generate the reference database, Symbiodinium OTU tables, and taxonomic assignments from the raw sequence data. Downstream statistical analysis and figure generation is then performed in the R/analysis.R script.

*Code for the bioinformatic pipeline utilized here has been generalized to be readily used on new datasets at http://github.com/jrcunning/SymITS2

Repository contents:

Makefile:

• This Makefile codes the entire bioinformatic pipeline utilized in this study with calls to scripts in the Shell/ and R/ directories. Given the raw sequence reads (data/Cunning_3967Raw10232015.zip), the list of samples contained therein (data/fastq_list.txt), and the list of accession numbers to include in the reference database (data/accn_nos.txt), execution of this Makefile using make will generate OTUs, build the ITS2 reference database, and assign taxonomy using 100% clustering, 97% clustering, and 97%-within-sample clustering.

Shell/:

• fetch_seqs.sh: This script downloads sequence data from NCBI given a list of named sequences with accession numbers (e.g., >C1_AF333515)
• merge_qc_reads.sh: This script unzips the fastq archive, merges and QC's reads, filters out chimeras, and trims primer sequences.
• otus_100.sh: This script clusters ITS2 sequences at 100% identity across all samples.
• otus_97.sh: This script clusters ITS2 sequences at 97% identity across all samples.
• otus_97_bysample.sh: This script separates ITS2 sequences by sample and clusters at 97% identity within each sample independently.
• prep_ITS2db.sh: This script prepares the ITS2 reference database from downloaded sequences by aligning sequences within clades, trimming to equal length, and removing duplicated sequences.
• run_blast_poormatches.sh: This script identifies OTU representative sequences with poor matches to the ITS2 reference database (<90% similarity to best match) and BLASTs these sequences against the NCBI nt database.

R/:

• analysis.R: This script codes all of the analysis downstream of OTU clustering and taxonomic assignment, including OTU quality filtering, summary statistics, and analysis of Symbiodinium community composition using multivariate statistics and network analysis. This script also generates all of the figures presented in the published manuscript (figures/Fig*.png).
• functions.R: This script contains several accessory functions that are utilized in the analysis.R script.
• run_nw.R: This script assigns taxonomy to all OTUs by performing Needleman-Wunsch global alignments of representative sequences against the ITS2 reference database.

data/:

Primary data:

• Cunning_3967Raw20232015.zip: Archive of raw sequencing reads in fastq format.
• coastline/: Contains data downloaded from the NOAA GSHHG database used to create map figures.
• EDNAFULL: Nucleotide substitution matrix utilized in global alignment for taxonomic assignments.
• STJ_envdata2.csv: Contains SST and chlorophyll a data for north and south shores of St. John
• accn_nos.txt: The list of named Symbiodinium ITS2 sequences with accession numbers (e.g., >C1_AF333515) to be included in the reference database.
• fastq_list.txt: The names of the fastq files in which paired reads for each sample can be found (needed for merging paired reads).
• mapping_file.txt: Metadata (e.g. species, collection site) for each sample in the dataset.

Intermediate data (these files are all created by executing make):

• ITS2db_trimmed.fasta: The trimmed ITS2 reference database
• ITS2db_trimmed_derep.fasta:: The trimmed and dereplicated ITS2 reference database
• ITS2db_trimmed_notuniques_otus.txt: A list of the non-unique sequences in the ITS2 reference database
• otus_100/: OTUs, representative sequences, and taxonomic assignments generated by 100% clustering
• otus_97/: OTUs, representative sequences, and taxonomic assignments generated by 97% clustering across samples
• otus_97_bysample/: OTUs, representative sequences, and taxonomic assignments generated by 97%-within-sample-clustering

figures/:

• Fig*.png: Each of the figures presented in the published manuscript. The code that produces each of these figures is in R/analysis.R.

supp/:

• STJ2012_supp.Rmd: R Markdown document containing all supplemental information and analysis.
• STJ2012_supp.html: Output of supplemental R Markdown document.

Software dependencies:

The following software is utilized by components of this code:

• GNU parallel
• illumina-utils
• oligotyping
• usearch
• cutadapt
• QIIME
• blast
• R (see session info below)

R > sessionInfo()

R version 3.3.1 (2016-06-21)
Platform: x86_64-apple-darwin13.4.0 (64-bit)
Running under: OS X 10.12.3 (Sierra)

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats4    parallel  stats     graphics  grDevices utils     methods   base     

other attached packages:
 [1] Biostrings_2.40.2   XVector_0.12.1      IRanges_2.6.1       S4Vectors_0.10.3   
 [5] BiocGenerics_0.18.0 pBrackets_1.0       RColorBrewer_1.1-2  stringr_1.1.0      
 [9] igraph_1.0.1        reshape2_1.4.1      multcompView_0.1-7  vegan_2.4-1        
[13] lattice_0.20-34     permute_0.9-4       rgdal_1.1-10        sp_1.2-3           
[17] phyloseq_1.16.2