⚠️ Work In Progress

This pipeline currently contains bugs that will give inaccurate results.

It is only suitable for testing and development until those bugs are fixed.

marple-pgt

Pipeline for analysis of Puccinia graminis f. sp. tritici genomic reads

Install

Before installing, ensure the licences of miniconda and software dependencies in env.yml are compatible with your usage.

The installer:

• Installs conda (miniconda)
• Installs dependencies from eny.yml into the environment marple-pgt

./install/install.sh

For WSL, you should keep the marple-pgt directory in the Linux file sytem (e.g. ~/marple-pgt), and not the windows file system (e.g. /mnt/c/Users/me/Documents/marple-pgt) as you may get an error:

OSError: [Errno 40] Too many levels of symbolic links

Tutorial

Let's go through the pipeline using a fictional example.

In this example, 2 samples have already been sequenced (using nanpore sequencing) and basecalled using Guppy.

Our starting point is a directory for each sample containing the fastq files concatenated together after being created by Guppy.

reads directory
example/cz2/cz2.fastq
example/uk20/uk20.fastq

1. Reset the example and navigate to the example directory

   ./reset_example.sh
   cd example

2. Align the reads for each sample to the reference genes, extract the CDS, and create a report

   ../src/reads_to_cds_concat.sh --trim no uk20/uk20.fastq cz2/cz2.fastq 

   The --trim no is used to skip adapter trimming by Porechop to save time for the example.

3. Inspect the report by opening example/multiqc_report.html in your browser.

4. Use the extracted and concatenated CDS of the new samples to create a tree. For the example create a very small tree (takes ~5 minutes) by running:

   ../src/cds_concat_to_tree_imgs.sh \
       --start ../data/12_samples_276_genes_cds.fa.gz \
       --out_dir tree \
       --name 2_new_samples \
       */*_cds_concat.fasta

   If you were using real data, you could make the full tree by removing the --start parameter like so. This can take hours to run.

   ../src/cds_concat_to_tree_imgs.sh \
       --out_dir tree \
       --name 2_new_samples \
       */*_cds_concat.fasta

5. Inspect the tree by opening example/tree/2_new_samples_country.pdf in your browser.

   Notice how country is shown as '?' for the new samples. We'll fix that in the next step.

6. Copy the metadata spreadsheet data/metadata_92.xlsx and rename it so that you have example\metadata_92_2_new_samples.xlsx.

   Add the new sample metadata to the spreadsheet so that the top 3 rows look like this:

   tree_name	tree_new_name	country	region	clade	accession	year	...
   cz2	cz2	Czechia	Europe			2013	...
   uk20	uk20	UK	Europe			2022	...

7. Use this metadata to make a new visualisation of the tree

   ../src/tree_to_imgs.sh \
       --meta metadata_92_2_new_samples.xlsx \
       --out_dir tree_with_new_metadata \
       tree/RAxML_bestTree.2_new_samples.newick

8. Inspect the new visualisation of the tree by opening example/tree_with_new_metadata/2_new_samples_country.pdf in your browser.

   The countries of the 2 new samples are now showing correctly.