CryptoGat201RNASeq

Cryptococcus neoformans RNA-seq Gat201 experiment in RPMI and RPMI + Serum media.

This repository is supplementary data, specifically RNA-seq dataset 2 data and analysis, accompanying the bioRxiv preprint:

A trade-off between proliferation and defense in the fungal pathogen Cryptococcus at alkaline pH is controlled by the transcription factor GAT201. Elizabeth S. Hughes, Laura R. Tuck, Zhenzhen He, Elizabeth R. Ballou, Edward W.J. Wallace. bioRxiv preprint, 2024 https://doi.org/10.1101/2023.06.14.543486

Analysis by Liz Hughes (liz.hughes@ed.ac.uk) and Edward Wallace (Edward.Wallace@ed.ac.uk), February 2021 through June 2024.

Data was created by Liz Hughes, December 2020. Raw data and gene counts are available in NCBI Gene Expression Omnibus GSE217345.

Contents

• src - scripts for nearly-raw data processing, including main script quantseqfwd.nf
• quantseqfwd_EH_050221 - results of quantseqfwd.nf
• quantseqfwd_test_output - practice run on subsampled data. Only useful for troubleshooting
• data_external - external data from published papers used for analysis
• Rmarkdown - scripts for more results-oriented data analysis and figures
• input_annotation - Cryptococcus genome annotation used for read alignment and counting.
• input_experiment - other input files including sample sheet
• results - output data and results

How we made the subsampled data

It is strongly recommended to do a test run on small yet real data - subsampled data - before running on a large dataset. An example of subsampling fastq files is:

gzip -dkc EH_050221_Data/1_S1_R1_001.fastq.gz \
  | head -n 400000 | gzip > EH_050221_Data_subsample/1_S1_R1_001_init100000.fastq.gz

gzip -dkc EH_050221_Data/2_S44_R1_001.fastq.gz \
  | head -n 400000 | gzip > EH_050221_Data_subsample/2_S44_R1_001_init100000.fastq.gz

How to run the pipeline

First edit the parameters in src/quantseqfwd.nf so that

• params.input_fq_dir points to an input directory containing all of your fastq files
• params.output_dir points to the output directory where you would like all the outputs stored.

Then run the command:

nextflow run src/quantseqfwd.nf -with-dag flowchart.png -with-report nextflow_report.html

The options -with-dag and -with-report are just to tell you a little more about the output, they are not strictly needed in order to run.