This pipeline can be used to generate the salmon quant files required by deseq2 for differential expression analysis.
A example shell script run template is provided named "run_template.sh"
The current version has been thinned out to only cover read trimming, QC reports, and quantification with salmon.
This is due to compatibility issues, other tools may be added back in future.
The typical command for running the pipeline is as follows:
nextflow run jambler24/bac_pangenome --reads sample_sheet.csv --genome refgenome.fa -profile ilifu
Mandatory arguments:
--reads Path to sample sheet
--genome Path to reference genome against which the reads will be aligned (in fasta format) for use in QC steps.
--gtf Path to the GTF formatted annotation file. Salmon does not work with mony of the gff formats.
--transcripts Path to the transcripts fasta file.
-profile Hardware config to use. Currently profile available for ilifu and UCT's HPC 'uct_hex' - create your own if necessary
Other arguments:
--outdir The output directory where the results will be saved
--SRAdir The directory where reads downloaded from the SRA will be stored
--email Set this parameter to your e-mail address to get a summary e-mail with details of the run sent to you when the workflow exits
-name
To allow for both local reads and reads from the SRA to be used, the pipeline has the ability to pull reads from the SRA based on the accession number (eg, SRR5989977).
The 'number' column must contain a unique value.
| number | origin | replicate | isolate | R1 | R2 |
|---|---|---|---|---|---|
| 1 | genomic | 1 | wgs_sample_1 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 2 | genomic | 2 | wgs_sample_1 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 3 | genomic | 3 | wgs_sample_1 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 4 | genomic | 1 | wgs_sample_2 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 5 | genomic | 2 | wgs_sample_2 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 6 | genomic | 3 | wgs_sample_2 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 7 | genomic | 1 | wgs_sample_3 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 8 | genomic | 2 | wgs_sample_3 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 9 | genomic | 3 | wgs_sample_3 | path/to/reads/reads_R1.fq | path/to/reads/reads_R2.fq |
| 10 | genomic | 1 | H37Rv | SRR5989977 |
In the above example, samples 1-9 are locally stored where sample 10 is a control sample from the SRA. Including the accession number in the R1 column will result in the reads from the SRA to be downloaded and used in the analysis. This must be exported to a csv file, with a comma ',' separating the columns:
number,origin,replicate,isolate,R1,R2
1,genomic,1,wgs_sample_1,path/to/reads/reads_R1.fq,path/to/reads/reads_R2.fq
2,genomic,2,wgs_sample_1,path/to/reads/reads_R1.fq,path/to/reads/reads_R2.fq
...
10,genomic,1,H37Rv,SRR5989977
Downstream analysis in R with Deseq2 requires a study design file.
The study design file is formatted like so:
run Unique_ID phenotype repeat
10 19119R-03-01 Wt 1
6 19119R-03-02 Wt 2
12 19119R-03-03 Wt 3
3 19119R-03-04 10X_DWD 1
8 19119R-03-05 10X_DWD 2
2 19119R-03-06 10X_DWD 3
7 19119R-03-07 1X_DWD 1
1 19119R-03-08 1X_DWD 2
4 19119R-03-09 1X_DWD 3
11 19119R-03-10 10X_GGT 1
5 19119R-03-11 10X_GGT 2
9 19119R-03-12 10X_GGT 3
Where the run column is the name of the output folder produced by salmon that contains the quant.sf files