Table of content

• Description
• Package installation
• Usage
  • 1.1. Retrieve fasta files from a general database
  • 1.2. Create the initial database
  • 1.3. Filter the sequences
  • 1.4. Create SA taxons
  • 2. Merge databases
• Qiime example
• References

Description

This package allow to create ASV specific reference databases from unfiltered FASTA files. FASTA file supported:

• ncbi: 'nt'
• ebi: 'ena'
• ddbj: 'arsa'
• RNAcentral
• UNITE
• Silva

It is mainly intended to be used by command line. However, some modules can be used inside python scripts. Most of the database creation is automatically done, only a few commands are left to the user.

CAUTION: this tool is not a pipeline but a set of modules.

Package installation

The package is available on pypi. It can be installed by the following command:

pip install asvmaker

Usage

ASVmaker is designed to be used by modules :

1.1. Retrieve fasta files from a general database

The first step involves downloading a FASTA file for a specific genus of interest from a general database : Silva, Unite, RNAcentral, ENA, NCBI or DDBJ. This file contains the genomic data necessary for subsequent analysis.

If you work from the Silva or UNITE fasta file, we recommand to extract genus sequences with grep -A 1 "Genus" filename.fasta. By this, ASVmaker will process your data quicker. CAUTION : fasta file sequences from Silva are multiligned. Use the following script to flatten the sequence on one single line : flatten.sh

# The genus of interest
GENUS=$1
# The name of the database. Pick among : ncbi,ebi,ddbj,rnaCentral,silva,unite
DB=$2
# Fasta file to proceed
SEQ=$3

1.2. Create the initial database

Next, ASVmaker enables the creation of a genus-specific database by using the downloaded FASTA file. Each sequence lineage is verified by accession number through the European Nucleotide Archive API if possible and through the NCBI Entrez API if the ENA one doesn't match. Users must specify primers to be used during the simulation of the amplification process, allowing for precise targeting of the desired genomic regions and ASV creation.

#  INIT DATABASE
python3 -m asvmaker \
    -inf database/${DB}/${GENUS}_${DB}_info_create.txt \
    create \
    -i ${SEQ} \
    -db ${DB} \
    -fp fw_primer.fasta \
    -rp rv_primer.fasta \
    -fmt 5 \
    -rmt 5 \
    -o database/${DB}/${GENUS}_${DB}_create.json

1.3. Filter the sequences

To enhance the quality and specificity of the analysis, ASVmaker provides the functionality to filter out redundant amplicons and exclude unwanted taxonomy. Redundant amplicons are ASVs sharing the same taxonomy. Unwanted taxonomy or species that are not of interest (e.g. : “sp.” or “aff.”) can also be filtered out, ensuring a more focused analysis on the target genus.

#  FILTER DATABASE
python3 -m asvmaker \
    -inf database/${DB}/${GENUS}_${DB}_info_filter.txt \
    filter \
    -i database/${DB}/${GENUS}_${DB}_create.json \
    -g1 ${GENUS} \
    -o database/${DB}/${GENUS}_${DB}_filter.json

#  EXPORT RESULTS
python3 -m asvmaker \
    -inf database/${DB}/${GENUS}_${DB}_info_exp1.txt \
    export \
    -i database/${DB}/${GENUS}_${DB}_filter.json \
    -sao database/${DB}/${GENUS}_${DB}_sa.txt 1

1.4. Create SA taxons

ASVmaker creates Shared Amplicon (SA) groups, which involve clustering identical ASVs which have different taxonomies. This grouping allows for a comprehensive understanding of the taxonomic diversity within the selected ASV, providing valuable precisions into the composition and dynamics of microbial communities.

#  EDIT DATABASE
python3 -m asvmaker \
    -inf database/${DB}/${GENUS}_${DB}_info_edit.txt \
    edit \
    -i database/${DB}/${GENUS}_${DB}_filter.json \
    -grp database/${DB}/${GENUS}_${DB}_sa_ext.txt \
    -o database/${DB}/${GENUS}_${DB}_edit.json

#  EXPORT RESULTS
python3 -m asvmaker \
    -inf database/${DB}/${GENUS}_${DB}_info_exp2.txt \
    export \
    -i database/${DB}/${GENUS}_${DB}_edit.json \
    -aop database/${DB}/${GENUS}_${DB}_asv.fasta

2. Merge databases

Moreover, ASVmaker offers the option to merge ASV specific databases from different general databases, providing flexibility to combine data from various sources. This merging process allows for a more comprehensive dataset, enabling comparative analysis and broader insights into the studied genus.

#  MERGE DATABASES
python3 -m asvmaker \
    -inf database/all/${GENUS}_info_merge.txt \
    merge \
    -i database/rnaCentral/${GENUS}_rnaCentral_edit.json \
    -sa1 database/rnaCentral/${GENUS}_rnaCentral_sa_ext.txt \
    -i2 database/unite/${GENUS}_unite_edit.json \
    -sa2 database/unite/${GENUS}_unite_sa_ext.txt \
    -o database/all/${GENUS}_merge.json

#  EXPORT RESULTS
python3 -m asvmaker \
    -inf database/all/${GENUS}_info_exp3.txt \
    export \
    -i database/all/${GENUS}_merge.json \
    -aop database/all/${GENUS}_asv.fasta

Qiime example

When an ASVmaker specific database has been created, it is possible to use it as a complementary database for Qiime2 ASV taxonomic identification.

Here, we present a method to use an ITS specific database created with ASVmaker on 38 fungus pathogen genera. This method involves a first identification with Qiime2 and a complementary step with the ASVmaker specific database.

In order to run such analysis, 3 files are required (+ FASTQ to analyse):

• A UNITE classifier : UNITE8.3_classifier.qza
• The ASVmaker ITS specific database phylo.fasta file : PathDB_BITS_2021.6-phylo.fasta
• A list of all the unique whole taxonomies for each genus in the ASVmaker specific database: Genus_Fungi_taxo.txt

A bash script (ITS.sh) makes a taxonomic attribution with Qiime2 and completes the identification with the ASVmaker specific database. Then, the results are exported as a table for each sample. If the detected genus is in the genera list, a column isPatho gives details about it. Columns description :

• #OTU ID : Qiime2 sequence identifier
• Reads Count : number of reads in the sample
• Relative Abundance : number of reads on total sum of reads in the sample
• Kingdom,Phylum Class, Order, Family, Genus, Species : UNITE taxonomy identification
• Unite Confidence : UNITE classifier identification confidence
• isPatho : taxon in pathogenes list
• Specific DB Taxonomy : taxonomy from ASVmaker specific database

Results table example :

#OTU ID	Reads Count	Relative Abundance	Kingdom	Phylum	Class	Order	Family	Genus	Species	Unite Confidence	isPatho	Specific DB Taxonomy
f369e7040d6a8ec809b813c86388be98	10696	0.2546545402599876	k__Fungi	p__Ascomycota	c__Sordariomycetes	o__Glomerellales	f__Plectosphaerellaceae	g__Plectosphaerella	s__Plectosphaerella_oratosquillae	0.9858436283038672	no	URS00007C0054_145972_Colletotrichum_pisi
21f34d539e27a351f59580aa39b530a9	7481	0.17811056616351603	k__Fungi	p__Ascomycota	c__Sordariomycetes	o__Glomerellales	f__Plectosphaerellaceae	g__Verticillium	s__unidentified	0.9981024689843628	yes	URS0002135E36_27337_Verticillium_SA1
c6e89cb4cb1459d20e8b29086b6579e0	10275	0.2446312080377125	k__Fungi	p__Ascomycota	c__Sordariomycetes	o__Glomerellales	f__Glomerellaceae	g__Colletotrichum	s__Colletotrichum_coccodes	0.9982691073091507	yes	URS00001143FA_27358_Colletotrichum_coccodes
23567ef88155066ea14693411a25e131	61	0.001452311794676444	k__Fungi	p__Ascomycota	c__Dothideomycetes	o__Pleosporales	f__Pleosporaceae	g__Alternaria	s__Alternaria_angustiovoidea	0.7681297203386827	yes	URS00002006C5_474922_Colletotrichum_gloeosporioides_complex
6cd9621117c8968bade94e1b2f69bd6b	385	0.009166230179515261	k__Fungi	p__Ascomycota	c__Sordariomycetes	o__Hypocreales	f__Hypocreales_fam_Incertae_sedis	g__Acremonium	s__Acremonium_furcatum	0.7784985803462298	no	URS00004BE307_27337_Verticillium_dahliae

References

Please cite : Plessis, C.; Jeanne, T.; Dionne, A.; Vivancos, J.; Droit, A.; Hogue, R. ASVmaker: A New Tool to Improve Taxonomic Identifications for Amplicon Sequencing Data. Plants 2023, 12, 3678. https://doi.org/10.3390/plants12213678