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## eDNA-seq Metabarcoding annotation pipeline
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**eDNA-seq Metabarcoding annotation** is a bioinformatics pipeline built using Snakemake, a workflow tool to run and manage tasks in any execution environment.
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## Introduction

**eDNA-seq Metabarcoding annotation** is specifically used for the analysis of environmental DNA metabarcoding NGS data, demultiplexing, filtering, dereplicating and taxonomic assignment (NCBI taxonomy and custom taxonomy).

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This pipeline has been initially tested with marine environmental DNA samples, using molecular markers such as Vert01 ([Riaz et al. 2011](https://doi.org/10.1093/nar/gkr732)), Teleo01 ([Valentini et al. 2016](https://doi.org/10.1111/mec.13428)), Chond01 or Mamm01 ([Taberlet et al. 2018](https://doi.org/10.1093/oso/9780198767220.001.0001)). The workflow should work with any organisms and environment. It is proven for large-scale data analysis.
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## Method


This workflow performs step by step analysis of metabarcoding data using [obitools](https://pythonhosted.org/OBITools/index.html).

The workflow assembles paired-end reads together (illuminapairedend), applies demultiplexing (ngsfilter), dereplicates sequences (obiuniq), detect and removes chimera and PCR clones (obiclean; obigrep) and assigns taxonomy to each representative sequences (NCBI taxonomy and custom taxonomy; ecotag). Ultimately, abundance distribution among samples of representative sequences is generated as a table.


## Workflow 


![workflow obitools](https://gitlab.mbb.univ-montp2.fr/edna/snakemake_only_obitools/-/raw/master/schema_only_obitools.png)

## Quick Start

See [Installion section](https://gitlab.mbb.univ-montp2.fr/edna/snakemake_rapidrun_obitools/-/wikis/Quickstart#installation) for installation instructions.


Download example data with:

```
curl -JLO http://gitlab.mbb.univ-montp2.fr/edna/snakemake_rapidrun_data_test/-/raw/master/test_rapidrun_data.tar.gz; tar zfxz test_rapidrun_data.tar.gz -C resources/test/
```

* `./resources/test/test_rapidrun_data/`: this folder contains a reference database for 4 markers (Teleo01; Mamm01; Vert01; Chond01), NGS metabarcoding raw data, required metadata to handle demultiplexing on RAPIDRUN format. 

If you have installed **eDNA-seq Metabarcoding annotation**, you can run the example data with:


```
snakemake --configfile config/config_test_rapidrun.yaml --cores 4 --use-conda
```

This will generate outputs into `./results` folder.

Final tables are available as:

```
results/projet1_chond_run1_ecotag_ncbi.csv   results/projet1_teleo_run5_ecotag_ncbi.csv   results/projet1_chond_run2_ecotag_ncbi.csv
results/projet1_teleo_run2_ecotag_ncbi.csv   results/projet1_vert_run3_ecotag_ncbi.csv    results/projet1_mamm_run4_ecotag_ncbi.csv
```

## Next steps

Now that you've gotten the example to work, see the [wiki](https://gitlab.mbb.univ-montp2.fr/edna/snakemake_rapidrun_obitools/-/wikis/Home) to navigate to the more detailed descriptions and instructions for exploring your own data.


## Software Requirements

* python3
* python3 required modules included: pandas, biopython, datrie, ConfigArgParse, appdirs, gitdb2, snakemake

## Credits

**eDNA-seq Metabarcoding annotation**  was coded and written by Virginie Marques and Pierre-Edouard Guerin.

We thank the following people for their help in the development of this pipeline: Agnes Duhamet, Alice Valentini, Apolline Gorry, Bastien Mace, David Mouillot, Emilie Boulanger, Laetitia Mathon, Laura Benestan, Stephanie Manel, Tony Dejean.


## Contributions and Support

:bug: If you are sure you have found a bug, please submit a bug report. You can submit your bug reports on Gitlab [here](https://gitlab.mbb.univ-montp2.fr/edna/snakemake_rapidrun_obitools/-/issues).
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For further information or help, don't hesitate to get in touch on Slack (you can join with this invite).

[![Get help on Slack](https://img.shields.io/badge/slack-cefebev%23metabarcoding_otu-4A154B?logo=slack)](https://cefebev.slack.com/archives/C01NYK8B9K7)