Introduction

sanger-tol/blobtoolkit is a bioinformatics pipeline that can be used to identify and analyse non-target DNA for eukaryotic genomes. It takes a samplesheet of BAM/CRAM/FASTQ/FASTA files as input, calculates genome statistics, coverage and completeness information, combines them in a TSV file by window size to create a BlobDir dataset and static plots.

1. Calculate genome statistics in windows (fastawindows)
2. Calculate Coverage (blobtk/depth)
3. Determine the appropriate BUSCO lineages from the taxonomy.
4. Run BUSCO (busco)
5. Extract BUSCO genes (blobtoolkit/extractbuscos)
6. Run Diamond BLASTp against extracted BUSCO genes (diamond/blastp)
7. Run BLASTx against sequences with no hit (diamond/blastx)
8. Run BLASTn against sequences still with not hit (blast/blastn)
9. Count BUSCO genes (blobtoolkit/countbuscos)
10. Generate combined sequence stats across various window sizes (blobtoolkit/windowstats)
11. Imports analysis results into a BlobDir dataset (blobtoolkit/blobdir)
12. Create static plot images (blobtk/images)

Usage

[!NOTE] If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

First, prepare a samplesheet with your input data that looks as follows:

samplesheet.csv:

csv sample,datatype,datafile,library_layout mMelMel3,hic,GCA_922984935.2.hic.mMelMel3.cram,PAIRED mMelMel1,illumina,GCA_922984935.2.illumina.mMelMel1.cram,PAIRED mMelMel3,ont,GCA_922984935.2.ont.mMelMel3.cram,SINGLE

Each row represents an aligned file. Rows with the same sample identifier are considered technical replicates. The datatype refers to the sequencing technology used to generate the underlying raw data and follows a controlled vocabulary (ont, hic, pacbio, pacbio_clr, illumina). The library layout indicates whether the reads are paired or single. The aligned read files can be generated using the sanger-tol/readmapping pipeline.

Now, you can run the pipeline using:

bash nextflow run sanger-tol/blobtoolkit \ -profile <docker/singularity/.../institute> \ --input samplesheet.csv \ --outdir <OUTDIR> \ --fasta genome.fasta \ --accession GCA_XXXXXXXXX.X \ --taxon XXXX \ --taxdump /path/to/taxdump/database \ --blastp /path/to/diamond/database \ --blastn /path/to/blastn/database \ --blastx /path/to/blastx/database

[!WARNING] Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

For more details, please refer to the usage documentation and the parameter documentation.

Pipeline output

For more details about the output files and reports, please refer to the output documentation.

Credits

sanger-tol/blobtoolkit was written in Nextflow by Alexander Ramos Diaz, Zaynab Butt, Matthieu Muffato, and Priyanka Surana. The orignal design and coding for BlobToolKit software and Snakemake pipeline was done by Richard Challis and Sujai Kumar.

We thank the following people for their assistance in the development of this pipeline:

• Guoying Qi
• Bethan Yates

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Citations

If you use sanger-tol/blobtoolkit for your analysis, please cite it using the following doi: 10.5281/zenodo.7949058

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.