ISeqDb 0.0.5

ISeqDb - Identify Sequences in Databases

ISeqDb - Identify Sequences in Databases

Screening of query dna sequences for the presence of specific target genes or proteins.

Query sequences can be metagenome assembles genomes (MAGs), contigs/scaffolds, multifasta and fasta files. Typically, databases contain multifasta homologous sequences. Databases are queried using megablast, blastn and blastx.

ISeqDd was tested to search for target genes or proteins in bacterial MAGs. ISeqDb was written primarily to search for the presence of genes encoding cyanotoxins and other genes encoding metabolites harmful to human health in cyanobacterial MAGs. Currently available databases obtained from NCBI (www.ncbi.nlm.nih.gov) include DNA sequences of genes encoding microcystins (mcyB, mcyD, mcyE), anatoxins (anaC, anaF), cylindrospermopsins (cyrJ), saxitoxins (sxtA, sxtI), and geosmin (geoA).

Any other database of homologous sequences can be used. This includes, for example, the rbcL gene used to classify diatoms (included). Additional nucleotide or protein databases may be created using the create_db module.

Installation

Requirements

python>=3.10, pandas, blast>=2.15, pip

conda/mamba should be already installed (https://mamba.readthedocs.io/en/latest/)

conda and pip

• Install dependencies creating a conda environment (through conda or mamba)

mamba create -y --name ISeqDb python>=3.10

mamba activate ISeqDb

mamba install -y python>=3.10 pip

mamba install -y blast>=2.15.0

• Install ISeqDb from PyPI

pip install ISeqDb

Uninstalling

To uninstall, or before installing new versions:

mamba deactivate

mamba env remove -y -n ISeqDb

Databases

Databases have a short description under their directory. New nucleotide and protein subject databases can be created using the create_db module. Databases work only with blast >=2.15 (after activating ISeqDb, check: blastn -version).

Basic usage

ISeqDb has three modules:

find_seqs: Identify sequences in databases using megablast (default), blastn or blastx ISeqDb find_seqs /path_to/queryfile.fasta /path_to/targetdatabase.tar.gz /path_to/outputfile.txt

inspect_db: Inspect or save the sequence database ISeqDb inspect_db /path_to/arch.tar.gz -- (inspect database) ISeqDb inspect_db /path_to/arch.tar.gz -d /outdir -- (inspect and save a copy arch.fasta)

create_db: Create a database from a fasta/multifasta file using makeblast ISeqDb create_db /path_to/arch.fasta -m "nucl" -- (nucleotide archives) ISeqDb create_db /path_to/arch.fasta -m "prot" -- (protein archives)

Usage

find_seqs

positional arguments:

queryfile path/file with extension .fasta .fas .fa .fna .ffn .faa; e.g. /dir/query.fasta

targetdb Database for the blast analysis (with path and file extension .tar.gz) - e.g. /dir/arch.tar.gz

outputfile Output name file (with path) - e.g. /dir/out.txt

options:

-h, --help show this help message and exit

-k {megablast,blastn,blastx}, --task {megablast,blastn,blastx} Task: megablast (nucl), blastn (nucl), blastx (prot); default=megablast

-m MAXTARGSEQ, --maxtargseq MAXTARGSEQ -Keep max target sequences >= maxtargseq; default=100

-e MINEVALUE, --minevalue MINEVALUE Keep hits with evalue >= minevalue; default=1e-6

-p MINPIDENT, --minpident MINPIDENT Keep hits with pident >= minpident (only megablast/blastn); default=85

-t THREADS, --threads THREADS Number of threads to use; default=1

-s {bitscore,pident,evalue,subject_seq_title}, --sortoutput {bitscore,pident,evalue,subject_seq_title} Sort output by colname; default=bitscore

-d {comma,semicolon,tab}, --delimiter {comma,semicolon,tab} Output delimiter: comma, semicolon, tab; default=comma

Output legend (in square brackets, the blast codes)

• query_id: query/sequence identifier [qacc]
• subject_accession: accession number or subject identifier in the database [sacc]
• pident: percentage of identical matches in query and subject sequences [pident]
• n_ident_match: number of identical bases/matches [nident]
• n_diff_match: number of different bases/matches [mismatch]
• n_gaps: total number of gaps [gaps]
• alignment_length: length of the alignment between query and subject sequences [length]
• query_start_al: start of alignment in query [qstart]
• query_end_al: end of alignment in query [qend]
• subj_start_al: start of alignment in subject [sstart]
• subj_end_al: end of alignment in subject [send]
• qlen: query sequence length [qlen]
• slen: subject sequence length [slen]
• bitscore: bit score [bitscore]
• evalue: expect value [evalue]
• subject_seq_title: title of subject sequence in database [stitle]
• align_query_seq: aligned part of query sequence [qseq]
• align_subj_seq: ligned part of subject sequence [sseq]

example:

ISeqDb find_seqs \

-k "megablast" -p 95 -e 1e-16 -t 8 -d tab -s pident \

/inputdir/file_nucleotide.fna \

/archdir/mcyE_2403.tar.gz \

/outdir/ISeqDb_output.txt

When comparing a DNA (nucleotide) fasta query with a protein sequence database, blastx must be used. The protein sequence database can be created using create_db with the option -m prot (see below).

inspect_db

positional arguments:

targetdb_inspect Database for the blast analysis (with path and file extension .tar.gz) - e.g. /dir/arch.tar.gz

options:

-h, --help show this help message and exit

-d SAVEDB, --savedb SAVEDB Output directory (with path) - e.g. /dir/; if not indicated, default=nosave

example:

ISeqDb inspect_db \

-d /outdir/ \

/archdir/mcyB_2403.tar.gz

create_db

positional arguments:

targetfasta path/file with extension .fasta .fas .fa .fna .ffn .faa; e.g. /dir/arch.fasta

options:

-h, --help show this help message and exit

-m {nucl,prot}, --moltype {nucl,prot} Molecule type in fasta file, nucl (nucleotide) or prot (protein)

example:

ISeqDb create_db \

-m prot \

/inputdir/geneseq.faa

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ISeqDb relies on BLAST®:

• BLAST® Command Line Applications User Manual [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2008-.
• Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. (2008) “BLAST+: architecture and applications.” BMC Bioinformatics 10:421. PubMed

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Acknowledgements

HORIZON-MSCA-2021-SE-01, AlgaeNet4AV, Project ID 101086437