nf_wochenende - a nextflow implementation of the Wochenende pipeline

See documentation on our Wiki at https://github.com/MHH-RCUG/nf_wochenende/wiki

This is a portable version of the metagenomic alignment pipeline Wochenende which uses Nextflow. This should allow most users to use Wochenende more easily and efficiently.

Wochenende runs alignment of short reads (eg Illumina) or long reads (eg Oxford Nanopore) against a reference sequence. It is relevant for genomics and metagenomics. Wochenende is simple (python script), portable and is easy to configure with a central config file.

Features

Features include (see programs listed below at the bottom of this page)

• QC (Fastqc)
• pre alignment duplicate removal (perldup)
• pre alignment poor sequence removal (Prinseq - used for single ended reads only)
• trimming (Trimmomatic or fastp or trim galore or ea-utils)
• alignment (bwa mem, or minimap2 for short reads, minimap2 or ngmlr for long reads)
• SAM-> BAM conversion (samtools and sambamba)
• Report % aligned reads (samtools)
• Output unmapped reads as fastq (samtools) (from v1.4)
• Post-alignment duplicate removal (Samtools from v1.7.8, Sambamba)
• Removal reads with x mismatches (bamtools), adjustable from v1.7.3
• Realignment (Abra2)
• MD tag marking (Samtools)
• Normalization (to Bacteria per Human cell, RPMM Reads Per Million sequenced reads per Million reference bases etc, see Reporting section in the docs for details)
• Visualization (chromosome coverage, intended for bacteria in metagenomics projects) (from v1.4)
• Growth rate estimation. Estimate how fast detected bacteria are growing
• Rare species prediction (Raspir)

Project Haybaler https://github.com/MHH-RCUG/haybaler allows postprocessing of Wochenende results:

• collation/integration of multiple reports (reporting csv or bam.txt files) using Python Pandas
• prepare results for heatmaps
• create heatmaps using multiple different R libraries

Did you know ?

Wochenende means weekend in German. The original developer, Tobias, called the pipeline Wochenende, because you can start it running and go off to enjoy your weekend early (at least, that was the plan!).

Preprint

Please view and cite the Wochenende preprint at https://www.biorxiv.org/content/10.1101/2022.03.18.484377v2

Paper

The paper is now in BMC genomics at https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-022-08985-9

Reference sequences

See the Wiki section Installation on where to download the reference sequence. If you work with clinical data, ie. where the main contaminant is human, that you should NOT remove the human from this sequence, since this will lead to false positive assignments of human reads to various bacteria. Reads are short, and the aligner will try to assign the human reads to bacteria (with mismatches) which can cause problems. If you work with other contaminants, eg mouse associated metagenomes, then you will need to remove the human and add the mouse genome to the supplied reference sequence (see the Wiki section on Building a reference sequence).

Installation

Quick install and run guide for nf_wochenende

• See full installation docs here: https://github.com/MHH-RCUG/nf_wochenende/wiki

• Clone the repo, get the fasta reference sequence

• Edit config in config.yaml

• Edit config in nextflow.config (cluster scheduler or local server, path to Wochenende and haybaler git clones and conda envs)

• Get a small read fastq file as input (present in the repo for testing)

• Edit start_nf.sh

• Run bash start_nf.sh

Output

• By default in output/wochenende
• One subfolder is output for each successful stage (nextflow process)
• See docs here: https://github.com/MHH-RCUG/nf_wochenende/wiki

Note that if you want to avoid Nextflow for some reason you can just run the Python script - python run_Wochenende.py

Tools used

• Alignerboost. GPL3, in dependencies folder.
• ABRA2
• bamtools
• BWA
• ea-utils
• fastp
• FastQC
• FastUniq
• Minimap2
• NGMLR
• perldup Already copied to dependencies folder with permission. Developed by Richard Leggett.
• PRINSEQ
• sambamba
• samtools
• trim_galore
• trimmomatic

Postprocessing

• Haybaler

Optional extras

• growth rate
• raspir

Nextflow version authors

• Colin Davenport
• Lisa Hollstein
• Ilona Rosenboom

Questions ?

Please post an issue in this repo.