qiime2-nf

Microbiome analysis with QIIME2

Getting started

1. Install nextflow, either directly or with conda

2. Install one of docker, singularity, or conda

3. In a new directory, write your configurations to the file nextflow.config. See Example config.

4. Execute the workflow

   nextflow run -resume Liulab/qiime2-nf

For more details on running nextflow, run nextflow help.

Using slurm

Simply tell nextflow to use the slurm profile. Multiple profiles can be specified together, separated by a comma. For example:

nextflow run -resume Liulab/qiime2-nf -profile docker,slurm

Beocat

On KSU's Beocat, singularity and slurm are available. It's recommended to use the provided profiles:

nextflow run -resume Liulab/qiime2-nf -profile singularity,slurm

Input

The input is expected to be a pair of gziped fastq for the forward and reverse read sequences, and a text file listing the samples barcode.

Default location

By default, the pipeline will look for fastq files with R1 and R2 in their name under the data/ subdirectory (relative to the current working directory). It will also look for any file with the .txt extension to use as the barcodes file.

You can either prepare your data according to this default or change where to find the data with the reads and barcode parameters. See Optional pipeline parameters.

Barcode format

The barcode file contain 4 tab-separated columns. These are the string barcode, the forward sequence, the reverse sequence, and the sample ID, respectively. For example:

barcode	GGATCGTAATAC	GATTATCGACGA	1AA
barcode	GGTTATTTGGCG	GTCGTGTAGCCT	1AB
barcode	CGTGATCCGCTA	ATCGCACAGTAA	1AE

Configuration

Required parameters

There are two steps in the pipeline that require parameters based on the results of previous steps. Initially, when these parameters are unspecified, the pipeline will halt, reporting an error. This is expected, just add the required parameter(s) and rerun the pipeline (make sure to specify -resume). The pipeline will proceed from where it stopped previously.

See "Moving Pictures" tutorial for how to pick an appropriate value for these parameters. The relevant output files will be found in v11nDir (default: out/visualization), and rawDir (default: out/raw-data).

For Denoising

Two parameters are required for this step

• truncF is the position at which the forward sequences should be truncated due to a drop-off in quality.
• truncR is the position at which the reverse sequences should be truncated due to a drop-off in quality.

For Core Metrics Analysis

One parameter (samplingDepth) is required during the alpha and beta diversity analysis process. This is the total frequency that each sample should be rarefied to prior to computing the diversity metrics.

Optional QIIME2 parameters

Some of the underlying QIIME2 plugins also take optional parameters. Below is a list of the parameter name to use for each step and where to find documentation for these params.

• demultiplexing: demuxExtra cutadapt docs
• summarizing demultiplexed sequences: demuxSumExtra demux docs
• denoising: denoiseExtra dada2 docs
• visualizing the denoisig stats: visualizeDenoiseStatsExtra metadata docs
• building phylogenetic trees: phylogenyExtra phylogeny docs
• classifying taxonomy: taxonomyClassificationExtra feature-classifier docs
• visualizing taxonomy: visualizeTaxonomyExtra metadata docs

Optional pipeline parameters

• reads: wildcard pattern to look for input reads (default: "data/*R{1,2}*.fastq{,.gz}")
• barcode: pattern to look for the barcode file (default: "data/*.txt")
• classifier: ML model to use for taxonomy classification (default: https://data.qiime2.org/2019.7/common/gg-13-8-99-515-806-nb-classifier.qza)
• prefix: prefix to add to output files (default: name of the current working directory)
• outdir: output directory
• v11nDir: QIIME2 visualizations directory (default: ${outdir}/visualization)
• rawDir: raw data from QIIME2 artifacts directory (default: ${outdir}/raw-data)

Multithreading

Some tasks in the pipeline can make use of multiple cores. These are tagged with the label multithreaded inside the main pipeline script. By default, these processes run with a single core. In order to allocate more cores, add the following snippet to nextflow.config, replacing 4 with the number of cores to use.

process { withLabel: multithreaded { cpus = 4 } }

Example config

/* nextflow.config */
params {
    // Customize where to find the input
    reads = "sample-r{1,2}.fq.gz"
    barcode = "barcode.txt"
    // Customize prefix for output files
    prefix = "my-awesome-project"
    classifier = "https://data.qiime2.org/2019.7/common/silva-132-99-nb-classifier.qza"
    // Extra parameter for qiime
    taxonomyClassificationExtra = "--p-confidence 0.8"
    truncF = 200
    truncR = 210
    samplingDepth = 5000
}
process {
    // Set processes tagged with multithreaded to use more threads
    withLabel: multithreaded {
        cpus = 12
    }
    // Customize resource for a specific step
    withName: buildPhylogeneticTrees {
        memory = 32.GB
    }
}
// Change where to save conda environment(s)
conda.cacheDir = "/home/bob/nextflow-conda-envs"
// Send a notification email when the pipeline terminates
notification.enabled = true
notification.to = "bob@example.com"

See Configuration for more details.

Output

QIIME2 produces two types of output artifact (.qza) and visualization (.qzv). You can either interact with these files using the QIIME2 provided CLI, and artifact API.

Underneath, these files are really just zip archives that contains the data and some additional metadata/information. You can use unzip to unpack the file and inspect its content directly.

Common Issues

Nextflow fails to download file from an https source with the error javax.net.ssl.SSLHandshakeException: Received fatal alert: handshake_failure

1. Download the Java Cryptography Extension(JCE) zip file for Java 8 from here.
2. Uncompress the downloaded archive
3. Move local_policy.jar and US_export_policy.jar to $JAVA_HOME/jre/lib/security. If there is no jre/ subdirectory under $JAVA_HOME/, move to $JAVA_HOME/lib/security instead

Credits

qiime2-nf was originally written by Ha Le and Jake Carlson.