16S_QIIME2

This is a Snakemake based 16S QIIME2 pipeline.

Installation

To install, we assume you already have installed Miniconda3 (4.7.10+) (https://docs.conda.io/en/latest/miniconda.html)

• Clone the repository:

git clone https://github.com/junglee0713/16S_QIIME2.git

• Create a conda environment:

cd 16S_QIIME2
conda env create -f environment.yml

• The following software also need to be installed:
  • dnabc (https://github.com/PennChopMicrobiomeProgram/dnabc)
  • unassigner (https://github.com/kylebittinger/unassigner)

To run the pipeline, activate the envrionment (currently based on QIIME2 2019.7) by entering source activate qiime2-2019.7 (Make sure to install dnabc and unassigner in the qiime2-2019.7 envrionment)

Required input files for the pipeline

To run the pipeline, we need

• Multiplexed R1/R2 read pairs (Undetermined_S0_L001_R1_001.fastq.gz, Undetermined_S0_L001_R2_001.fastq.gz), and
• QIIME2 compatible mapping file
  • Tab delimited
  • The first two columns should be SampleID (or #SampleID) and BarcodeSequence

How to run

• Create a project directory, e.g. /home/leej39/16S_QIIME2/test and put the mapping file, e.g. test_mapping_file.tsv in the project directory
• Edit config.yml so that it suits your project. In particular,
  • all: project: path to the project directory, e.g. /home/leej39/16S_QIIME2/test
  • all: mux_dir: the direcotry containing multiplexed R1/R2 read pairs, e.g. /home/leej39/16S_QIIME2/test/multiplexed_fastq
  • all: mapping: the name of mapping file, e.g. test_mapping_file.tsv
• To run the pipeline, activate the envrionment by entering source activate qiime2-2019.7, cd into 16S_QIIME2 and execute e.g.

snakemake \
    --configfile path/to/config.yml \
    --keep-going \
    --latency-wait 90 \
    --notemp \
    --printshellcmds

• When submitting jobs using qsub, you may run e.g. bash run_snakemake.bash path/to/config_test.yml
  • bash dryrun_snakemake.bash path/to/config_test.yml for dryrun
  • bash unlock_snakemake.bash path/to/config_test.yml for unlocking

Intermediate steps and corresponding input/output

Demultiplexing

Input

• Multiplexed R1/R2 read pairs
• QIIME2 compatible mapping file

Output

• Demultiplexed fastq(.gz) files
• Total read count summary (tsv)
• QIIME2 compatible manifest file (csv)

QIIME2 import

Input

• QIIME2 compatible manifest file
• Demultiplexed fastq files

Output

• QIIME2 PairedEndSequencesWithQuality artifact and corresponding visualization
• QIIME2-generated demultiplexing stats

DADA2 denoise

Input

• QIIME2 PairedEndSequencesWithQuality artifact

Output

• Feature table (QIIME2 artifact, tsv)
• Representative sequences (QIIME2 artifact, fasta)

Taxonomy classification

Input

• Representative sequences

Output

• Taxonomy classification table (QIIME2 artifact, tsv)

Tree building

Input

• Representative sequences

Output

• Aligned sequence
• Masked (aligned) sequence
• Unrooted tree
• Rooted tree

Diversity calculation

Input

• Rooted tree

Output

• Various QIIME2 diversity metric artifacts
• Faith phylogenetic diversity vector (tsv)
• Weighted/unweighted UniFrac distance matrices (tsv)

Unassigner

Input

• Representative sequences (fasta)

Output

• Unassigner output (tsv) for species level classification of representative sequences

dada2_species

Input

• Representative sequences (fasta)

Output

• Dada2 species assignments (tsv)
• Dada2 Raw data for loading in R (RData format)

vsearch

Input

• Representative sequences (fasta)

Output

• Vsearch report (tsv) customized to be like BLAST results (see config.yml)
• Vsearch list of representative sequences that aligned (fasta)

Basic Bioinformatics Report

Input

• QIIME2 compatible mapping file and output from diversity calculation

Output

• Basic Bioinformatics Report containging heatmap, relative proportion bar graph, alpha diversity plots, beta diversity plots, and per sample read counts in HTML format.