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workingwithchewBBACA.md

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From assemblies to cgMLST profiles: working with chewBBACA

Task 1 - Organizing and fetching the data

# Get chewBBACA
docker pull mickaelsilva/chewbbaca
 
# Create a working folder and enter it
mkdir test_chewbbaca
cd test_chewbbaca
 
# Create folders to store the genomes

mkdir complete_genomes
mkdir other_assemblies
 
# create a list of complete genomes to download
# we will be using nano for text editing
touch 2down_cg.txt
nano 2down_cg.txt
 
# copy and paste this list of links into the file (to exit nano press ctrl+x and save the changes with "y" and enter)
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/007/265/GCF_000007265.1_ASM726v1/GCF_000007265.1_ASM726v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/012/705/GCF_000012705.1_ASM1270v1/GCF_000012705.1_ASM1270v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/196/055/GCF_000196055.1_ASM19605v1/GCF_000196055.1_ASM19605v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/427/035/GCF_000427035.1_09mas018883/GCF_000427035.1_09mas018883_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/001/026/925/GCF_001026925.1_ASM102692v1/GCF_001026925.1_ASM102692v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/689/235/GCF_000689235.1_GBCO_p1/GCF_000689235.1_GBCO_p1_genomic.fna.gz
 
# create a list of assembled genomes to download
touch 2down_other.txt
nano 2down_other.txt
 
# copy and paste this list of links into the file
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/167/715/GCF_000167715.1_ASM16771v1/GCF_000167715.1_ASM16771v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/001/694/615/GCF_001694615.1_ASM169461v1/GCF_001694615.1_ASM169461v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/001/694/415/GCF_001694415.1_ASM169441v1/GCF_001694415.1_ASM169441v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/002/246/945/GCF_002246945.1_ASM224694v1/GCF_002246945.1_ASM224694v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/736/485/GCF_000736485.1_ASM73648v1/GCF_000736485.1_ASM73648v1_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/221/325/GCF_000221325.1_ASM22132v2/GCF_000221325.1_ASM22132v2_genomic.fna.gz
ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/287/955/GCF_000287955.1_ASM28795v1/GCF_000287955.1_ASM28795v1_genomic.fna.gz

# download and decompress the complete genomes files
cd complete_genomes
wget -i ../2down_cg.txt
gunzip ./*.gz
ls
cd ..
 
# download and decompress other assemblies
cd other_assemblies
wget -i ../2down_other.txt
gunzip ./*.gz
ls
cd ..

Task 2 - Create a schema based on the downloaded complete genomes

# start a container with chewBBACA
docker run -it -v ~/:/data/ mickaelsilva/chewbbaca bash
 
# go to our working folder
cd /data/test_chewbbaca/
 
# run chewBBACA to create the schema using the complete genomes
chewBBACA.py CreateSchema -i complete_genomes/ -o schema_seed --cpu 8 -t "Streptococcus agalactiae"

Work assignment - Create a schema based on YOUR downloaded complete genomes

# start a container with chewBBACA
docker run -it -v ~/media/volume/mydata:/mydata/ mickaelsilva/chewbbaca bash

# go to your working folder
cd /mydata/

# make sure the genomes you will be using are on unix (assemblies made with innuca don't need this step)

dos2unix /mydata/my/genomes/folder/*

# create your own training file based on the reference genome you want
prodigal -i /mydata/reference_genome.fasta -t mytrainingFile.trn -p single

#run the schema creation with your specific training file
chewBBACA.py CreateSchema -i /mydata/complete_genomes/ -o schema_seed --cpu 6 --ptf mytrainingFile.trn

Task 3 - Run an allele call using the created schema and the genomes available


# create a file with full paths of the genomes we want to call alleles
 
find /data/test_chewbbaca/complete_genomes/* >listgenomes.txt
find /data/test_chewbbaca/other_assemblies/* >>listgenomes.txt
find /path/to/innuca/assemblies/*.fasta >>listgenomes.txt
 
# open listgenomes.txt and check if thereis 1 line with full paths for each assembly (20 in total).Press "q" to exit less after the check
less listgenomes.txt
 
# use the created list of genomes to call the alleles 
chewBBACA.py AlleleCall -i listgenomes.txt -g schema_seed/ -o results --cpu 8 -t "Streptococcus agalactiae"

Work assignment - Run an allele call using the created schema with YOUR samples and the genomes YOU have

# start a container with chewBBACA if you have exited the last one and cd to your data folder
docker run -it -v ~/media/volume/mydata:/mydata/< mickaelsilva/chewbbaca bash
cd /mydata/

# create a file with full paths of the genomes you want to call alleles
 
find /mydata/complete_genomes/* >listgenomes.txt
find /mydata/my_other_assemblies/* >>listgenomes.txt
 
# open listgenomes.txt and check if there is 1 line with full paths for each assembly. Press "q" to exit less after the check
less listgenomes.txt

# use the created list of genomes to call the alleles 
chewBBACA.py AlleleCall -i listgenomes.txt -g schema_seed/ -o results --cpu 8 --ptf mytrainingFile.trn

Task 4 - test genome allele call quality


# at the end of the command the program will try to open the html. Press "q" and "y" to exit that action when prompted
chewBBACA.py TestGenomeQuality -i results/<results_20171207T150515>/results_alleles.tsv -o testcq -n 12 -s 5 -t 300

# download testcq/GenomeQualityPlot.html to your local computer and open with your favorite browser

Task 5 - Extract the profile for phyloviz

# Extract a profile suitable to be used by phyloviz

chewBBACA.py ExtractCgMLST -i results/<results_20171207T150515>/results_alleles.tsv -o my_cgMLST -r results/<results_20171207T150515>/RepeatedLoci.txt -p 0.95

# download my_cgMLST/cgMLST.tsv to your computer and use it later for phyloviz

#create a metadata file on your computer
FILE	species	ST	contigs
GCF_000007265.1_ASM726v1_genomic.fna	sagalactiae	110	1
GCF_000012705.1_ASM1270v1_genomic.fna	sagalactiae	7	1
GCF_000196055.1_ASM19605v1_genomic.fna	sagalactiae	23	1
GCF_000427035.1_09mas018883_genomic.fna	sagalactiae	1	1
GCF_000689235.1_GBCO_p1_genomic.fna	sagalactiae	17	1
GCF_001026925.1_ASM102692v1_genomic.fna	sagalactiae	1	1
GCF_000167715.1_ASM16771v1_genomic.fna	sagalactiae	-	553
GCF_000221325.1_ASM22132v2_genomic.fna	sagalactiae	67	8
GCF_000736485.1_ASM73648v1_genomic.fna	sagalactiae	7	8
GCF_001694415.1_ASM169441v1_genomic.fna	sagalactiae	196	493
GCF_001694615.1_ASM169461v1_genomic.fna	sagalactiae	-	553
GCF_002246945.1_ASM224694v1_genomic.fna	sagalactiae	7	5
GCF_000287955.1_ASM28795v1_genomic.fna	sagalactiae	-	18
ERR048560.contigs.length_GCcontent_kmerCov.mappingCov.polished.fasta	?	?	?
ERR840705.contigs.length_GCcontent_kmerCov.mappingCov.polished.fasta	?	?	?
SRR1790752.contigs.length_GCcontent.mappingCov.polished.fasta	?	?	?
SRR3320580.contigs.length_GCcontent_kmerCov.mappingCov.polished.fasta	?	?	?
SRR4414149.contigs.length_GCcontent_kmerCov.mappingCov.polished.fasta	?	?	?
SRR5575009.contigs.length_GCcontent_kmerCov.mappingCov.polished.fasta	?	?	?
SRR5575010.contigs.length_GCcontent_kmerCov.mappingCov.polished.fasta	?	?	?

Task 6 - schema eval

chewBBACA.py SchemaEvaluator -i schema_seed/ -l rms/Myschema.html --cpu 8

zip -r myschemaEval.zip rms/

#download the myschemaEval.zip to your computer, unzip it and open the Myschema.html with firefox

Extra - create prodigal training file

prodigal -i assembly.fasta -t tfName.trn -p single

Extra - create schema with a personalized prodigal training file

chewBBACA.py CreateSchema -i genomes/ -o schema_seed --cpu 6 --ptf tfName.trn

Extra - get information about each locus

uniprot_find.py -i schema_seed/ -t proteinID_Genome.tsv
#creates a file new_protids.tsv with the information per locus and respective name on uniprot and a link to the sequence