README.md

Marine Omics d2s Pipeline

Rapidly generate a distance matrix between samples based on shared kmers in raw reads. This pipeline takes raw fastq files as input and will calculate d2s (a normalised distance metric) based on shared kmers between all pairs of samples. If target and background genomes are provided, reads will be filtered to include reads that map to the target and that don't map to background. The pipeline is ideally suited to clustering samples based on kmer profiles of reads from algal symbionts (family Symbiodiniaceae)

Input data can be from any type of sequencing (RNAseq, WGS, Radseq) but should be the same type across all samples.

graph TD;
	fastq-->bwa;
	target_genome-->bwa;
	background_genome-->bwa;
	bwa-->samtools_fastq;
	samtools_fastq-->jellyfish;
	jellyfish-->d2ssect;
	samtools_fastq-->d2ssect;

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Installation

First install and configure nextflow. See here for instructions specific to JCU machines (zodiac, genomics1, genomics2)

Run a test to make sure everything is working. This test should work on a system with singularity installed.

nextflow run marine-omics/mod2s -profile singularity,test_pe -r main

Quick Start

As a minimum, mod2s requires a set of raw read data (fastq files). Assuming you have raw data paths in a file named samples.csv you would run an analysis with;

nextflow run marine-omics/mod2s -profile zodiac -r main --samples samples.csv --outdir myout

Note the profile here is zodiac which will load predefined settings for the JCU HPC. Other alternatives include genomics or a custom profile that you create yourself with -c custom.config.

If you provide a target genome via the --target_ref argument, mod2s will calculate d2s statistics based only on reads that map to the target;

nextflow run marine-omics/mod2s -profile zodiac -r main --samples samples.csv --target_ref symbiont.fasta --outdir myout

If you provide both a target (--target_ref) and background ref (--background_ref) only reads that map to the target AND that do not map to background will be used.

nextflow run marine-omics/mod2s -profile zodiac -r main --samples samples.csv --target_ref symbiont.fasta --background_ref host.fasta --outdir myout

Raw Data (samples.csv)

The paths to fastq files must be provided in csv format as in the example below;

sample,fastq_1,fastq_2
1,sample1_r1.fastq.gz,sample1_r2.fastq.gz
2,sample2_r1.fastq.gz,sample2_r2.fastq.gz

See here for more detail on the samples.csv format.

Reference sequences

Reference sequences should be provided in fasta format.

If you are working with coral sequences you might be unsure of the correct reference to use for symbiont targets. A good place to start is to run the moqc pipeline which should give you an idea of the symbiont genus that is most dominant. In the most common case this will be Cladocopium in which case a good choice for the reference sequence is the transcriptome available from reefgenomics.

Outputs

Successful completion of the pipeline will produce outputs in the <outdir> you provided including;

- *d2s* : Matrices of d2s distances for all pairs of samples. 

# something in here to make an MDS plot