hundo 1.1.5

Amplicon processing protocol

• Performs quality control based on quality, can trim adapters, and remove sequences matching a contaminant database

• Handles paired-end read merging

• Integrates de novo and reference-based chimera filtering

• Clusters sequences and annotates using databases that are downloaded as needed

• Generates standard outputs for these data like a newick tree, a tabular OTU table with taxonomy, and .biom.

This workflow is built using Snakemake and makes use of Bioconda to install its dependencies.

Documentation

For complete documentation and install instructions, see:

https://hundo.readthedocs.io

Install

This protocol leverages the work of Bioconda and depends on conda. For complete setup of these, please see:

https://bioconda.github.io/#using-bioconda

Really, you just need to make sure conda is executable and you’ve set up your channels (numbers 1 and 2). Then:

conda install python=3.6 pyyaml snakemake biopython biom-format=2.1.5
pip install hundo

Usage

Running samples through annotation requires that input FASTQs be paired-end, named in a semi-conventional style starting sample ID, contain “_R1” (or “_r1”) and “_R2” (or “_r2”) index identifiers, and have an extension “.fastq” or “.fq”. The files may be gzipped and end with “.gz”. By default, both R1 and R2 need to be larger than 10K in size. This cutoff is arbitrary and can be set using --prefilter-file-size.

Using the example data of the mothur SOP located in our tests directory, we can annotate across SILVA using:

cd example
hundo annotate \
    --filter-adapters qc_references/adapters.fa.gz \
    --filter-contaminants qc_references/phix174.fa.gz \
    --out-dir mothur_sop_silva \
    --database-dir annotation_references \
    --reference-database silva \
    mothur_sop_data

Dependencies are installed by default in the results directory defined on the command line as --out-dir. If you want to re-use dependencies across many analyses and not have to re-install each time you update the output directory, use Snakemake’s --conda-prefix:

hundo annotate \
    --out-dir mothur_sop_silva \
    --database-dir annotation_references \
    --reference-database silva \
    mothur_sop_data \
    --conda-prefix /Users/brow015/devel/hundo/example/conda

Output

OTU.biom

Biom table with raw counts per sample and their associated taxonomic assignment formatted to be compatible with downstream tools like phyloseq.

OTU.fasta

Representative DNA sequences of each OTU.

OTU.tree

Newick tree representation of aligned OTU sequences.

OTU.txt

Tab-delimited text table with columns OTU ID, a column for each sample, and taxonomy assignment in the final column as a comma delimited list.

OTU_aligned.fasta

OTU sequences after alignment using Clustal Omega.

all-sequences.fasta

Quality-controlled, dereplicated DNA sequences of all samples. The header of each record identifies the sample of origin and the count resulting from dereplication.

blast-hits.txt

The BLAST assignments per OTU sequence.

summary.html

Captures and summarizes data of the experimental dataset. Things like sequence quality, counts per sample at varying stages of pre-processing, and summarized taxonomic composition per sample across phylum, class, and order.