COI DB

Overview

This tool downloads sequences + metadata from GBIF and formats sequences of interest for use with downstream metabarcoding analyses.

Installation options

Option 1: Install with conda:

bash conda install -c bioconda coidb

Option 2: Download a release from the 'Releases' section, unpack it and then create and activate the conda environment. Finally, install the software:

bash conda env create -f environment.yml conda activate coidb python -m pip install .

Quick start

To see the steps that will be run, without actually running them, do:

bash coidb -n

Remove the -n flag to actually run the steps.

Output

The primary outputs of the tool are:

1. boldclusteredcleaned.fasta: A fasta file with sequences clustered at whatever threshold set in the config file (default is 1.0 which means 100% identity). The header of each sequence in this file has the format

```

GMGMN070-14 Animalia;Arthropoda;Insecta;Lepidoptera;Pieridae;Gonepteryx;Gonepteryx rhamni;BOLD:AAA9222 ```

In this example GMGMN070-14 is the representative id for the sequence and can be viewed in the BOLD database at https://www.boldsystems.org/index.php/Public_RecordView?processid=GMGMN070-14.

1. bold_clustered.sintax.fasta: This fasta file is compatible with the SINTAX classification tool implemented in vsearch and has headers with the format:

```

GMGMN070-14;tax=d:Animalia,k:Arthropoda,p:Insecta,c:Lepidoptera,o:Pieridae,f:Gonepteryx,g:Gonepteryx rhamni,s:BOLD:AAA9222 ```

[!NOTE] In the SINTAX formatted headers, the taxonomic ranks are shifted to allow classification down to BOLD_bin. Since SINTAX only allows for ranks prefixed with 'd' (for domain) 'k' (kingdom), 'p' (phylum), 'c' (class), 'o' (order), 'f' (family), 'g' (genus), or 's' (species) we shift the taxonomy so that kingdom becomes domain, etc., and prefix the BOLD bin id with 's'.

1. boldclustered.assigntaxonomy.fasta and boldclustered.addSpecies.fasta: These fasta files are compatible with the assignTaxonomy and addSpecies functions implemented in DADA2. For the assignTaxonomy file the headers have the format:

```

Animalia;Arthropoda;Insecta;Lepidoptera;Pieridae;Gonepteryx;Gonepteryx rhamni; ```

and for the addSpecies file the headers have the format:

```

GMGMN070-14 Gonepteryx rhamni ```

Configuration

There are a few configurable parameters that modifies how sequences are filtered and clustered. You can modify these parameters using a config file in yaml format. The default setup looks like this:

yaml database: # url to download info and sequence files from url: "https://hosted-datasets.gbif.org/ibol/ibol.zip" # gene of interest (will be used to filter sequences) gene: - "COI-5P" # phyla of interest (omit this in order to include all phyla) phyla: [] # Percent identity to cluster seqs in the database by pid: 1.0

Gene types

By default, only sequences named 'COI-5P' are included in the final output. To modify this behaviour you can supply a config file in yaml format via -c <path-to-configfile.yaml>. For example, to also include 'COI-3P' sequences you can create a config file, e.g. named config.yaml with these contents:

yaml database: gene: - 'COI-5P' - 'COI-3P'

Then run coidb as:

bash coidb -c config.yaml

Phyla

The default is to include sequences from all taxa. However, you can filter the resulting sequences to only those from one or more phyla. For instance, to only include sequences from the phyla 'Arthropoda' and 'Chordata' you supply a config file with these contents:

yaml database: phyla: - 'Arthropoda' - 'Chordata'

Clustering

After sequences have been filtered to the genes and phyla of interest they are clustered on a per-species (or BOLD BIN id where applicable) basis using vsearch. By default this clustering is performed at 100% identity. To change this behaviour, to e.g. 95% identity make sure your config file contains:

yaml database: pid: 0.95

Command line options

The coidb tool is a wrapper for a small snakemake workflow that handles all the downloading, filtering and clustering.

``` usage: coidb [-h] [-n] [-j CORES] [-f] [-u] [-c [CONFIG_FILE ...]] [--cluster-config CLUSTER_CONFIG] [--workdir WORKDIR] [-p] [-t] [targets ...]

positional arguments: targets File(s) to create or steps to run. If omitted, the full pipeline is run.

options: -h, --help show this help message and exit -n, --dryrun Only print what to do, don't do anything [False] -j CORES, --cores CORES Number of cores to run with [4] -f, --force Force workflow run -u, --unlock Unlock working directory -c [CONFIGFILE ...], --config-file [CONFIGFILE ...] Path to configuration file --cluster-config CLUSTER_CONFIG Path to cluster config (for running on SLURM) --workdir WORKDIR Working directory. Defaults to current dir -p, --printshellcmds Print shell commands -t, --touch Touch output files (mark them up to date without really changing them) instead of running their commands. ```

How it works

Firstly sequence and taxonomic information for records in the BOLD database is downloaded from the GBIF Hosted Datasets. GBIF processes taxonomic information from BOLD in order to resolve ambiguous assignments for BOLD BINs. When there are conflicting assignments at a taxonomic rank an 80% consensus rule is applied to keep e.g. a species level assignment if four out of five names in the BIN are equal Kõljalg et al 2020. This data is then filtered to only keep records annotated as 'COI-5P' and assigned to a BIN ID and duplicate entries are removed.

Taxonomy

The taxonomic information obtained from GBIF is then parsed in order to extract species names to BOLD BINs. This is done by: 1. find all BOLD BINs with a taxonomic assignment at genus level, these likely have species names assigned from GBIF (see methods for species assignment here) 2. obtain all parent taxonomic ids for BOLD BINs from step 1 and use these to look up the species name for the BOLD BINs. 3. For BOLD BINs where species name look-up failed in step 2, try to obtain species name using the GBIF API.

The taxonomic data is then searched for rows where missing values for ranks are filled with the last known higher level rank, suffixed with _X. For instance,

| BOLD BIN | kingdom | phylum | class | order | family | genus | species | |--------------|-----------|-----------------|-------|-------------|--------|-------|---------| | BOLD:ACX1129 | Animalia | Platyhelminthes | NaN | Polycladida | NaN | NaN | NaN | | BOLD:ACX6548 | Chromista | Ochrophyta | NaN | NaN | NaN | NaN | NaN |

becomes:

| BOLD BIN | kingdom | phylum | class | order | family | genus | species | |--------------|-----------|-----------------|-------------------|---------------|----------------|-----------------|------------------| | BOLD:ACX1129 | Animalia | Platyhelminthes | PlatyhelminthesX | Polycladida | PolycladidaX | PolycladidaXX | PolycladidaXXX | | BOLD:ACX6548 | Chromista | Ochrophyta | OchrophytaX | OchrophytaXX | OchrophytaXXX | OchrophytaXXXX | Ochrophyta_XXXXX |

As you can see, an X is appended for each downstream rank with a missing assignment.

BOLD BINs are then screened for cases where there are more than 1 unique parent lineage for the same taxonomic assignment. For example, the following taxonomic information may be found for BOLD BINs with assignment 'Aphaenogaster' at the genus level.

| kingdom | phylym | class | order | family | genus | |----------|------------|-------------|---------------|---------------|---------------| | Animalia | AnimaliaX | AnimaliaXX | AnimaliaXXX | AnimaliaXXXX | Aphaenogaster | | Animalia | Arthropoda | Insecta | Hymenoptera | Formicidae | Aphaenogaster |

A check is first made to see if unique parent lineages can be obtained by removing BINs that only have missing assignments for parent ranks up to and including phylum. If that doesn't result in a unique parent lineage, the conflicting rank assignments are prefixed with the lowest assigned parent rank.

For example, BOLD BINs with genus level assignment 'Paralagenidium' have both k_Chromista;p_Oomycota;c_Peronosporea;o_Peronosporales;f_Pythiaceae and k_Chromista;p_Ochrophyta;c_Ochrophyta_X;o_Ochrophyta_XX;f_Ochrophyta_XXX as parent lineages. Since these conflicts cannot be resolved by removing BINs (all BINs have assignments at phylum level), the taxa labels at genus and species level are prefixed with either Pythiaceae_ or Ochrophyta_XXX_.

Sequence processing

Sequences are then processed to remove gap characters and leading and trailing Ns. After this, any sequences with remaining non-standard characters are removed. Sequences are then clustered at 100% identity using vsearch (Rognes et al. 2016). This clustering is done separately for sequences assigned to each BIN ID.

Step-by-step

You can also run the coidb tool in steps, e.g. if you are only interested in some of the files or if you want to inspect the results before proceeding to the next step. This is done using the positional argument targets.

Valid targets are download, filter and cluster.

Step 1: Download

For example, to only download files from GBIF you can run:

bash coidb download

This should produce two files bold_info.tsv and bold_seqs.txt containing metadata and nucleotide sequences, respectively.

Step 2: Filter

To also filter the bold_info.tsv and bold_seqs.txt files (according to the default 'COI-5P' gene or any other genes/phyla you've defined in the optional config file) you can run:

bash coidb filter

This filters sequences in bold_seqs.txt and entries in bold_info.tsv to potential genes and phyla of interest, respectively. Entries are then merged so that only sequences with relevant information are kept. Output files from this step are bold_filtered.fasta and bold_info_filtered.tsv.

Step 3: Clustering

The final step clusters sequences in bold_filtered.fasta on a per-species basis. This means that for each species, the sequences are gathered, clustered with vsearch and only the representative sequences are kept. In this step sequences can either have a species name or a BOLD BIN ID (e.g. BOLD:AAY5017) and are treated as being equivalent.

To run the clustering step, do:

bash coidb cluster

The end result is a file bold_clustered.fasta.

Step 4: Clean headers

The clean step removes extra information from sequence headers generated as part of clustering. To run this step, do:

bash coidb clean

Step 5: Generate SINTAX/DADA2 formatted fasta

To also get the SINTAX and/or DADA2 formatted fasta file, do:

bash coidb format_sintax

or

bash coidb format_dada2