Introduction

EMC-CanMic is a WORK IN PROGRESS bioinformatics pipeline that helps with analyzing the microbiome within RNA sequencing data, obtained from humans. As input it requires a samplesheet and paired-end FASTQ files, it performs quality control and trimming on the reads, filters out reads mapping to a human reference genome and taxonomically classifies the remaining reads. As output, you receive all intermediate outputs as well as a BIOM file with the classifications and a MultiQC report of the QC metrics and tools used.

The steps performed by the EMC-CanMic pipeline are shown in the figure below:

And include the following:

1. Quality control (Fastp)
2. Filter out reads mapping to GRCh38 (BWA-MEM2 and Samtools)
3. Summarize mapping statistics (Samtools)
4. Convert SAM file to FASTQ (Samtools)
5. Taxonomic classification (Kraken2)
6. Re-estimation of abundances (Bracken)
7. Convert Kreport to BIOM (Kraken-biom)
8. Generate report with quality metrics and used tools (MultiQC)

Installation

To use EMC-CanMic on your machine, clone this GitHub repository.

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:

sample,fastq_1,fastq_2
CONTROL_1, BR_PVP_0705_R1.fastq.gz, BR_PVP_0705_R2.fastq.gz

Each row represents a pair of fastq files.

Now, you can run the EMC-CanMic pipeline using:

nextflow run emc-cancermicro \
   -profile <docker/singularity/conda/.../institute> \
   --input samplesheet.csv \
   --outdir <OUTDIR> \
   --fasta <path/to/reference_genome_fasta> \
   --kraken2_db <path/to/kraken2_database/directory/>

Change the default "null" values in "nextflow.config" to the paths you will use most often to save time. Values in this file will be overwritten by the values specified in the command.

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.

Credits

EMC-CanMic was originally written by Birgit Rijvers.

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

• Willem de Koning

Support

If you have questions or issues while using this pipeline, please create an issue in this repository.

Citations

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.