Bayesian LCA-based Taxonomic Classification Method (BLCA) is a Bayesian-based method that provides a solid probabilistic basis for evaluating the taxonomic assignments for the query sequences with bootstrap confidence scores, which is based on Bayesian posterior probability that quantitatively weigh each database hit sequence according to its similarity to the query sequence - the more similar database hit sequence to the query, the more its contribution to the taxonomic assignment of the query. We implemented the above algorithm as a python3 package at https://github.com/qunfengdong/BLCA.

• Python (version 3 or above)
• Linux

• libyaml

• python3-dev
• setuptools

• Biopython
• pyyaml

• BLAST binary (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.3.0/)
• MUSCLE (http://www.drive5.com/muscle/downloads.htm)

• 16S Microbial database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/16SMicrobial.tar.gz)

• If you intend to use customized database, please make sure the sequence headers are of the format below wherein the XXXXXXX should be replaced with GI numbers. This will help us with obtaining the taxonomical classification.

>gi|XXXXXXX|gb|YYYYYYYY|

• Also format the database using BLAST suite

Checkout the source code: https://github.com/qunfengdong/BLCA

$ git clone https://github.com/qunfengdong/BLCA.git
$ cd BLCA
$ python3 setup.py install

$ cd BLCA/example

Update the paths to BLAST executable directory, BLAST database and Muscle binary file

BLAST_BINARY = 'path/to/dir/ncbi-blast-2.3.0+/bin'
BLAST_DATABASE = 'path/to/file/16SMicrobial'
MUSCLE_BINARY = '/path/to/bin/muscle'

Run BLCA

$ python3
>>> import blca
>>> import config
>>> blca.execute()

Output is stored in file called annotation_output.txt, open the file in Excel or text editor.

Go to any directory or folder, and download the example FASTA file and config file to the same folder. Example FASTA file is available at https://raw.githubusercontent.com/qunfengdong/BLCA/master/example/example_input_file.fasta Example config.py file is available at https://raw.githubusercontent.com/qunfengdong/BLCA/master/example/config.py

$ pwd
/path/to/current_directory
$ wget https://raw.githubusercontent.com/qunfengdong/BLCA/master/example/example_input_file.fasta
$ https://raw.githubusercontent.com/qunfengdong/BLCA/master/example/config.py

Description of the parameters in the config file "config.py"

[current_directory]$ cat config.py
##-----------------------------------------------------------
## Input file in FASTA format
FILENAME = 'example_input_file.fasta'

## Output file
OUTFILE = 'annotation_output.txt'

## BLAST parameters
BLAST_BINARY = 'path/to/dir/ncbi-blast-2.3.0+/bin'
## Path to the actual indexed BLAST database with 16SMicrobial.n* files
BLAST_DATABASE = 'path/to/file/16SMicrobial'
## MUSCLE executable file
MUSCLE_BINARY = '/path/to/bin/muscle'

## BLAST output filtering options
## Select hits with at least a score 100
BLAST_CUTOFF_SCORE = 100
## Select hits within less then 10% score of the top hit
BLAST_CUTOFF_PERCENT = 10
## Select hits with at least 95% coverage
BLAST_COVERAGE = 95
## Select hits with at least 95% percentage identity
BLAST_PERCENTAGE_IDENTITY = 95

## Mutiple sequence alignment parameters
## Select 10 base pairs of hit sequences
## upstream and downstream of the alignment
HIT_SEQUENCE_BPS = 10

## Alignment scoring values
ALIGNMENT_GAP = -2.5
ALIGNMENT_MISMATCH = -2
ALIGNMENT_MATCH = 1

## Bootstrap
BOOTSTRAP = 100
##-----------------------------------------------------------

[current_directory]$ python3
>>> import blca
>>> import config
>>> blca.execute()

import blca
import config
blca.execute()

Execute the file 'analysis.py' at the terminal

[current_directory]$ python3 analysis.py

• A text file with sequence id in the first column, taxonomy annotation with confidence scores in the parentheses.
• Each taxonomy level is separated by tab.
• If no taxonomy annotation is available, it is listed as 'NA'

sequence_id  superkingdom	phylum	class	order	family	genus	species subspecies  organism
seq1  Bacteria (100)  Firmicutes (100)  Bacilli (100) Bacillales (100)  Bacillaceae (100) Bacillus (100)  Bacillus subtilis (60)  Bacillus subtilis subsp. spizizenii (43)  Bacillus subtilis subsp. spizizenii (24) 
seq2  Bacteria (100)  Proteobacteria (100)  Gammaproteobacteria (100) Pseudomonadales (100) Pseudomonadaceae (100)  Pseudomonas (100) Pseudomonas mucidolens (21) Pseudomonas cedrina subsp. fulgida (6)  Pseudomonas mucidolens (21)

If you put them in Excel, they should be in the following order.

Column A: sequence_id
Column B: superkingdom
Column C: phylum
Column D: class
Column E: order
Column F: family
Column G: genus
Column H: species
Column I: subspecies
Column J: organism

• Make sure the sequence headers in the FASTA file DO NOT have "|" (pipe) characters in them. Please change them to "_" (underscore)

• Version 0.6 first public release

• Dr. Xiang Gao, theoretical conception and algorithm development
• Dr. Qunfeng Dong, algorithm development
• Kashi Revanna, program coding and package development
• Huaiying Lin, program coding and testing

GNU

• BLAST program: Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., & Madden T.L. (2008) "BLAST+: architecture and applications." BMC Bioinformatics 10:421.
• MUSCLE: Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput.Nucleic Acids Res. 32(5):1792-1797. doi:10.1093/nar/gkh340
• Biopython: Cock PA, Antao T, Chang JT, Bradman BA, Cox CJ, Dalke A, Friedberg I, Hamelryck T, Kauff F, Wilczynski B and de Hoon MJL (2009) Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics, 25, 1422-1423