Finishing the basic workflow

Overview

Questions

• What does the full sample workflow look like?
• How can we report some initial results from this analysis?

Objectives

• Finish the sample workflow to produce a final MultiQC report
• See more ways to handle awkward software by adding extra setup shell commands

For reference, this is the Snakefile you should have to start the episode.

We’ve seen how to link rules in a pipeline and how to merge all the results at the final step. This is the basic pattern for many analysis workflows. For simplicity, in episode 5, we just used the cat command to combine all the .count files but now we’ll use MultiQC to combine the results from Kallisto and FastQC into a single report for all samples. We’ll also add in an alternative quantification tool called Salmon and this will complete the pipeline.

The full workflow we are constructing

---

• fastq_quality_trimmer is part of the FastX toolkit and removes low-quality basecalls from the raw reads. We first used it in episode 2.
• FastQC calculates a variety of metrics on a FASTQ file and produces an HTML report and a ZIP file. We introduced this in episode 6.
• Kallisto aligns the reads to a reference transcriptome and produces a table of transcript abundance. We first used it in episode 3.
• Salmon is a alternative to Kallisto, using a different alignment algorithm. We’ve not used it yet.
• MultiQC combines the reports from various tools, including FastQC, Kallisto, and Salmon, into a single HTML report over all samples. This is by no means a full RNA-Seq analysis report but today it completes our Snakemake pipeline.

At this point we have everything we need, in terms of Snakemake knowledge, to add the two remaining tools and complete the Snakefile. As with FastQC, some quirks in the way the new tools work will need to be accounted for.

Adding Salmon as an alternative to Kallisto

---

At the end of the previous episode we modified the kallisto rule by declaring that the output of the rule was a directory, rather than explicitly listing the three files that Kallisto writes in that directory. You should ensure that you are working with this new version of the kallisto rule as it will be a template for adding a salmon rule.

Adding Salmon as an alternative to Kallisto

An alternative application for transcript quantification is Salmon. The procedure is virtually identical, to Kallisto, having an indexing step and a quantification step. Note that in real usage you are advised to prepare and add decoy sequences to the transcriptome index, but for the purposes of this tutorial we’ll just keep things as simple as possible.

Based upon the following command templates:

BASH

$ salmon index -t <transcriptome as fasta> -i <index name> -k 31
$ salmon quant -i <index name> -l A -1 <fastq1> -2 <fastq2> --validateMappings -o <output path>

Add a pair of rules to index and align the reads with Salmon. Note that:

1. Unlike Kallisto, the index produced by Salmon is a directory of files, not a single file - so both of these new rules will produce a directory as output.
2. As noted in the last episode, you only need the directory() marker on the outputs of rules, not the inputs.

Show me the solution

rule salmon_quant:
    output: directory("salmon.{sample}")
    input:
        index = "Saccharomyces_cerevisiae.R64-1-1.salmon_index",
        fq1   = "trimmed/{sample}_1.fq",
        fq2   = "trimmed/{sample}_2.fq",
    shell:
        "salmon quant -i {input.index} -l A -1 {input.fq1} -2 {input.fq2} --validateMappings -o {output}"

rule salmon_index:
    output:
        idx = directory("{strain}.salmon_index")
    input:
        fasta = "transcriptome/{strain}.cdna.all.fa.gz"
    shell:
        "salmon index -t {input.fasta} -i {output.idx} -k 31"

If you copied the kallisto_index rule and logged the output of salmon_index to a file this is fine. Just make sure when copying and pasting that you change all the parts that need to change!

Combining the outputs with MultiQC

---

MultiQC scans for analysis report files in a given directory and all subdirectories, then makes a report of everything it finds. It knows about FastQC, Salmon and Kallisto outputs so we should be able to compile a report on all these. To try this out and scan the current directory, simply run:

BASH

$ multiqc . -o multiqc_out

Adding a MultiQC rule

Earlier, in episode 5, we made a basic summary-type rule called all_counts. Now make a multiqc rule that gathers up all the FastQC, Salmon and Kallisto reports.

Considerations:

1. Your rule is going to have several named inputs, and these inputs will be lists of files generated with expand() functions.
2. Ensure that both kallisto_quant and salmon_quant are run on all 9 samples, that is all three repeats of all three conditions.
3. Run FastQC on the untrimmed reads only, and note that MultiQC specifically uses the .zip files for input, not the .html.
4. Since multiqc scans for input files, the input names don’t have to be explicitly mentioned in the shell part.

Show me the solution

rule multiqc:
    output: directory('multiqc_out')
    input:
        salmon =   expand("salmon.{cond}_{rep}", cond=CONDITIONS, rep=REPLICATES),
        kallisto = expand("kallisto.{cond}_{rep}", cond=CONDITIONS, rep=REPLICATES),
        fastqc =   expand("reads.{cond}_{rep}_{end}_fastqc.zip", cond=CONDITIONS, rep=REPLICATES, end=["1","2"]),
    shell:
        "multiqc . -o multiqc_out"

Since the rule has no wildcards, you can run it by giving either the rule name or the output directory name as a target.

BASH

$ snakemake -j1 -p multiqc

...or equivalently...

$ snakemake -j1 -p multiqc_out

Fixing Kallisto

You may notice that MultiQC is not capturing any Kallisto output when making the reports. The reason for this is given in the MultiQC manual here:

Note - MultiQC parses the standard out from Kallisto, not any of its output files (abundance.h5, abundance.tsv, and run_info.json). As such, you must capture the Kallisto output to a file when running it for use with MultiQC.

Fix the Snakefile so that Kallisto terminal output is redirected to a file and can be collected by MultiQC.

• Hint 1: The manual above is not quite right - you need to capture both stdout and stderr, so use >& rather than >, as we did with the indexing step.
• Hint 2: MultiQC does not mind what you call the file, so choose your own sensible name.

Show me the solution

# Kallisto quantification of one sample, with log capture.
rule kallisto_quant:
    output: directory("kallisto.{sample}")
    input:
        index = "Saccharomyces_cerevisiae.R64-1-1.kallisto_index",
        fq1   = "trimmed/{sample}_1.fq",
        fq2   = "trimmed/{sample}_2.fq",
    shell:
        """mkdir {output}
           kallisto quant -i {input.index} -o {output} {input.fq1} {input.fq2} >& {output}/kallisto_quant.log
        """

There are several perfectly good ways of structuring this, so don’t worry if your answer is different.

A gotcha with the above version is that the output directory needs to be created before kallisto quant is run, much like with FastQC. Remember that Snakemake deletes any existing outputs, including outputs that are directories, before the job is run, and while Kallisto will create the directory for you this is too late for the shell to make the log file and without the mkdir command it will report “No such file or directory”.

Another option is to make the log file outside of the directory, or stick with declaring the individual files as outputs, as when we first made the rule, in which case the directory will be made by Snakemake. It’s also possible to declare both the directory and a file within that directory as separate outputs, which is unusual but may be a good approach here.

Making the MultiQC rule more robust

Because MultiQC scans for suitable input rather than taking an explicit list of files, there is a risk that it picks up unwanted reports if you leave old files sitting in the directory. To make the rule fully explicit, one idea is to make a temporary directory and symlink all the files into it, and then tell MultiQC to look in there. Amend the multiqc rule so it does this.

• Hint 1: When making links, use ln -snr -t <target_dir> <src> to avoid link relativity issues.
• Hint 2: If you feel that tweaking some other rules would make this easier, feel free to do that.

Show me the solution

This solution will work with the version of kallisto_quant in the solution above.

rule multiqc:
    output:
        mqc_out = directory('multiqc_out'),
        mqc_in  = directory('multiqc_in'),
    input:
        salmon =   expand("salmon.{cond}_{rep}", cond=CONDITIONS, rep=REPLICATES),
        kallisto = expand("kallisto.{cond}_{rep}", cond=CONDITIONS, rep=REPLICATES),
        fastqc =   expand("reads.{cond}_{rep}_{end}_fastqc.zip", cond=CONDITIONS, rep=REPLICATES, end=["1","2"]),
    shell:
        """mkdir {output.mqc_in}
           ln -snr -t {output.mqc_in} {input}
           multiqc {output.mqc_in} -o {output.mqc_out}
        """

To see the actual MultiQC report, open the file multiqc_out/multiqc_report.html in a web browser. You can do this directly from the command line, assuming you have a default browser configured.

On Linux environments:

BASH

$ xdg-open multiqc_out/multiqc_report.html

For MacOS:

BASH

$ open multiqc_out/multiqc_report.html

The report has a few issues, but we’ll not get distracted by the details of how to configure MultiQC to resolve them.

For reference, this is a Snakefile incorporating the changes made in this episode. You may now proceed to any later episode in the lesson using this workflow as a starting point.

Key Points

• Once a workflow is complete and working, there will still be room for refinement
• This completes the introduction to the fundamentals of Snakemake