Overview

zAMP is a Snakemake cli, written in Snaketool, for amplicon metagenomics anlysis. It includes state-of-the-art tools for performant and reproducibile analysis of 16S rRNA or ITS Illumina paired-end reads.

Starting from local fastq or SRA reads zAMP performs reads QC, ASV inference, taxonomic assignments and basic visualization plots. In addition, zAMP enables training taxonomic classifiers on specific primer-amplified regions in popular databases like greengenes2, SILVA and UNITE to increase sensitivity.

Installation

Dependencies

• Mamba
• Apptainer

from source

git clone https://github.com/metagenlab/zAMP.git
pip install -e zAMP

Usage

Prepare database

Greengenes2

• Download

wget http://ftp.microbio.me/greengenes_release/current/2022.10.backbone.full-length.fna.qza
wget http://ftp.microbio.me/greengenes_release/current/2022.10.backbone.tax.qza

• Export qza with qiime2 export

docker run -t -i -v $(pwd):/data quay.io/qiime2/tiny:2024.5 \
qiime tools export \
--input-path 2022.10.backbone.full-length.fna.qza \
--output-path greengenes2
docker run -t -i -v $(pwd):/data quay.io/qiime2/tiny:2024.5 \
qiime tools export \
--input-path 2022.10.backbone.tax.qza --output-path greengenes2

• Prepare database

zamp db --fasta greengenes2/dna-sequences.fasta \
--taxonomy greengenes2/taxonomy.tsv --name greengenes2 \
--fw-primer CCTACGGGNGGCWGCAG --rv-primer GACTACHVGGGTATCTAATCC \
-o greengenes2

Fungal ITS databases

Fungal ITS databases (Unite v10 and Eukaryome have been verified) do not contain the adjacent 18S/28S sequences (they contain 5.8S), where some of the commonly used PCR primers lie on. Extraction of the amplified region from the database would therefore not possible. It is important to adjust the cutadapt parameters so that only the lacking primer is not required. In the following example, we prepare a database for fungal ITS1 from Unite Db. In this case, the forward primer (lying of the 18S) will not be present in most sequences of Unite/Eukaryome (but the reverse primer lying on the 5.8S is present); therefore we set the forward primer as optional; the extracted sequences will start at the available 5' of the database and end at the reverse primer:

zamp db \
--fasta sh_refs_qiime_unite_ver10_dynamic_04.04.2024.fasta \
--taxonomy sh_taxonomy_qiime_unite_ver10_dynamic_04.04.2024.txt \
--name unite \
--fw-primer CYHRGYYATTTAGAGGWMSTAA --rv-primer RCKDYSTTCWTCRWYGHTGB \
--minlen 50 --maxlen 900 \
--cutadapt_args_fw "optional"

Run with prepared database

zamp run -i zamp/data/sra-samples.tsv \
-db greengenes2 \
--fw-primer CCTACGGGNGGCWGCAG --rv-primer GACTACHVGGGTATCTAATCC 

Evaluate database

The module zamp insilico allows you to insilico test a pair of PCR primers and evaluate their suitability to correctly amplify and classify taxons of interest. It also allows you to assess if your database is able to correctly determine the taxonomy of your species of interest, based on the expected amplicon.

To do so, the module performs an insilico PCR with your primer pair on a collection of publicly available assemblies (NCBI). The extracted amplicons are processed and classified by the main zAMP module and compared to the expected NCBI taxonomy. The output is a summary table indicating whether amplification occurs on the species of interest with the specified primer pair, how many amplicons are extracted from your query assemblies, and whether the taxonomy obtained with your database corresponds to the expected. As input, you need to provide the taxons to investigate as assembly accession names, tax IDs or queries, as well as the PCR primer pair and your database to evaluate (prepared by zamp db).

Example usage cases:

1. Using bacteria assembly accession names:

zamp insilico -i zamp/data/bacteria-accs.txt \
-db greengenes2 --accession \
--fw-primer CCTACGGGNGGCWGCAG --rv-primer GACTACHVGGGTATCTAATCC 

2. Using fungi tax IDs (requires additional ITS amplicon-specific parameters):

zamp insilico -i zamp/data/fungi-taxa.txt \
-db unite_db_v10 \ 
--fw-primer CYHRGYYATTTAGAGGWMSTAA --rv-primer RCKDYSTTCWTCRWYGHTGB \
--minlen 50 --maxlen 900

3. Using a query term. In this example, 100 assemblies will be downloaded per taxon (nb 100) including non-reference assemblies (not-only-ref):

zamp insilico -i "lactobacillus" \
-db ezbiocloud \
--fw-primer CCTACGGGNGGCWGCAG --rv-primer GACTACHVGGGTATCTAATCC \
--replace-empty -nb 100 --not-only-ref

Help

$ zamp -h
Usage: zamp [OPTIONS] COMMAND [ARGS]...

  Snakemake pipeline designed for convenient, reproducible and scalable
  amplicon-based metagenomics

  For more options, run: zamp command --help

Options:
  -v, --version  Show the version and exit.
  -h, --help     Show this message and exit.

Commands:
  db        Prepare database files for zAMP
  run       Run zAMP
  citation  Print zAMP and tools citations