Repository files navigation

nfcore/ampliseq is a bioinformatics analysis pipeline used for amplicon sequencing, supporting denoising of any amplicon and supports a variety of taxonomic databases for taxonomic assignment including 16S, ITS, CO1 and 18S. Phylogenetic placement is also possible. Multiple region analysis such as 5R is implemented. Supported is paired-end Illumina or single-end Illumina, PacBio and IonTorrent data. Default is the analysis of 16S rRNA gene amplicons sequenced paired-end with Illumina.

A video about relevance, usage and output of the pipeline (version 2.1.0; 26th Oct. 2021) can also be found inYouTube andbillibilli, the slides are deposited atfigshare.

The pipeline is built usingNextflow, a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker/Singularity containers making installation trivial and results highly reproducible. TheNextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies.

On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources. The results obtained from the full-sized test can be viewed on thenf-core website.

By default, the pipeline currently performs the following:

• Sequencing quality control (FastQC)
• Trimming of reads (Cutadapt)
• Infer Amplicon Sequence Variants (ASVs) (DADA2)
• Optional post-clustering withVSEARCH
• Predict whether ASVs are ribosomal RNA sequences (Barrnap)
• Phylogenetic placement (EPA-NG)
• Taxonomical classification using DADA2; alternatives areSINTAX,Kraken2, andQIIME2
• Excludes unwanted taxa, produces absolute and relative feature/taxa count tables and plots, plots alpha rarefaction curves, computes alpha and beta diversity indices and plots thereof (QIIME2)
• Creates phyloseq R objects (Phyloseq)
• Pipeline QC summaries (MultiQC)
• Pipeline summary report (R Markdown)

First, you need to know whether the sequencing files at hand are expected to contain primer sequences (usually yes) and if yes, what primer sequences. In the example below, the paired end sequencing data was produced with 515f (GTGYCAGCMGCCGCGGTAA) and 806r (GGACTACNVGGGTWTCTAAT) primers of the V4 region of the 16S rRNA gene. Please note, that those sequences should not contain any sequencing adapter sequences, only the sequence that matches the biological amplicon.

Next, the data needs to be organized in a folder, heredata, or detailed in a samplesheet (seeinput documentation).

Now, you can run the pipeline using:

nextflow run nf-core/ampliseq \   -profile<docker/singularity/.../institute> \   --input"data" \   --FW_primer"GTGYCAGCMGCCGCGGTAA" \   --RV_primer"GGACTACNVGGGTWTCTAAT" \   --outdir<OUTDIR>

For more details and further functionality, please refer to theusage documentation and theparameter documentation.

To see the results of an example test run with a full size dataset refer to theresults tab on the nf-core website pipeline page.For more details about the output files and reports, please refer to theoutput documentation.

nf-core/ampliseq was originally written by Daniel Straub (@d4straub) and Alexander Peltzer (@apeltzer) for use at theQuantitative Biology Center (QBiC) andMicrobial Ecology, Center for Applied Geosciences, part of Eberhard Karls Universität Tübingen (Germany). Daniel Lundin@erikrikarddaniel (Linnaeus University, Sweden) joined before pipeline release 2.0.0 and helped to improve the pipeline considerably.

We thank the following people for their extensive assistance in the development of this pipeline (in alphabetical order):

Adam Bennett,Diego Brambilla,Emelie Nilsson,Jeanette Tångrot,Lokeshwaran Manoharan,Marissa Dubbelaar,Sabrina Krakau,Sam Minot,Till Englert

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

For further information or help, don't hesitate to get in touch on theSlack#ampliseq channel (you can join withthis invite).

If you usenf-core/ampliseq for your analysis, please cite theampliseq article as follows:

Interpretations of Environmental Microbial Community Studies Are Biased by the Selected 16S rRNA (Gene) Amplicon Sequencing Pipeline

Daniel Straub, Nia Blackwell, Adrian Langarica-Fuentes, Alexander Peltzer, Sven Nahnsen, Sara Kleindienst

Frontiers in Microbiology 2020, 11:2652doi: 10.3389/fmicb.2020.550420.

You can cite thenf-core/ampliseq zenodo record for a specific version using the followingdoi: 10.5281/zenodo.1493841

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

You can cite thenf-core publication as follows:

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.