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Assembly

The assembly pipeline will assemble preprocessed reads into larger contigs, which can then be the basis for binning. Our pipeline does the following tasks:

  • Assembly with MEGAHIT
  • Contiguity check with Quast
  • Annotation of open reading frames with bakta

For long reads, assembly will be performed with Meta-Flye (see below).

Setup

As for every of these workflows, you will have to edit the params.yml file to specify the output location, database locations, and parameters (see the General setup page). Of importance for the assembly workflow is:

  • bakta database location.

Then, you can run the workflow like this:

nextflow run </path/to/this/repo>/workflows/assembly.nf \
	-c </path/to/this/repo>/config/run.config \
	-params-file params.yml \
	-with-trace -with-report

Input

The input for this process is a file containing the location of the preprocessed reads. This sample sheet file should look like this:

sampleID,forward,reverse,orphans
Sample_1,</path/to/reads/>reads_sample1_1.fq.gz,</path/to/reads/>reads_sample1_2.fq.gz,</path/to/reads/>reads_sample1_orphans.fq.gz
Sample_2,</path/to/reads/>reads_sample2_1.fq.gz,</path/to/reads/>reads_sample2_2.fq.gz,</path/to/reads/>reads_sample2_orphans.fq.gz

If you used the preprocessing workflow, you will not have to provide the sheet with the preprocessed reads. One output of this workflow is a file specifying the location of the preprocessed reads in the correct format and the assembly workflow will look for this file in the specific outdir location.

However, if you want to run assembly only on some samples or on samples processed in a different way, you can either specify the input parameter in the params.yml file or you can supply it when calling the nextflow process like this:

nextflow run </path/to/this/repo>/workflows/assembly.nf \
	-c </path/to/this/repo>/config/run.config \
	-params-file params.yml \
	-with-trace -with-report --input ./preprocessed_reads.csv

Output

This script will create the following outputs:

  • assembly: Main output folder, containing
    • megahit: Folder containing the megahit assemblies
    • bakta: Folder containing the output from bakta
    • quast_report.tsv: File tabulating the assembly quality for each sample
  • versions_assembly.yml: The versions for each of the tools using in the assembly pipeline

Long reads

For long reads, the input file with the cleaned reads looks different again.

sampleID,reads
Sample_1,</path/to/reads/>cleaned_reads_1.fq.gz
Sample_2,</path/to/reads/>cleaned_reads_2.fq.gz

Also, the long_reads parameter in the params.yml file needs to be set to true again.

Lastly, the file with the cleaned reads needs to be supplied as samples when running the nextflow command (otherwise, the assembly would run on the raw, unprocessed reads). If this info is stored in a file called cleaned_reads.csv, it would look like this!

nextflow run </path/to/this/repo>/workflows/assembly.nf \
	-c </path/to/this/repo>/config/run.config \
	-params-file params.yml \
	-with-trace -with-report --samples ./cleaned_reads.csv