genotyphi.py
Python 2.7.5+
SAMtools and BCFtools are also required if you are working from BAM files. Genotyphi has been tested with versions 1.1, 1.2, and 1.3 of both SAMtools and bcftools, subsequently we advise using the same version of both of these dependencies together i.e. SAMtools v1.2 and bcftools v1.2
Inputs are BAM or VCF files (mapped to the Typhi CT18 reference genome, AL513382.1).
For short read data, we recommend using the raw alignments (BAM files) as input (via --bam), since VCFs generated by different pipelines may have filtered out important data at the genotyping loci. Note you also need to supply the reference sequence that was used. If you don't have BAMs or you are really confident in the quality of your SNP calls in existing VCF files, you can provide your own VCFs (generated by read mapping) via --vcf.
For assemblies, we recommend using [ParSNP] and [HarvestTools] (http://harvest.readthedocs.org/) (version 1.0.1) to align genomes to the CT18 reference. The resulting multi-sample VCF file(s) can be passed directly to this script via --vcf_parsnp.
Generating input BAMS from reads (with example)
Generating input VCFs from assemblies (with example)
Note the BAM files must be sorted (e.g. using samtools sort)
python genotyphi.py --mode bam --bam *.bam --ref AL513382.fasta --ref_id AL513382.1 --output genotypes.txt
python genotyphi.py --mode vcf --vcf *.vcf --ref_id AL513382 --output genotypes.txt
Basic Usage - assemblies aligned with ParSNP (recommended if you only have assembly data available and no reads)
python genotyphi.py --mode vcf_parsnp --vcf parsnp.vcf --output genotypes.txt
-- mode Run mode, either bam, vcf or vcf_parsnp
Requires SAMtools and BCFtools
--bam Path to one or more BAM files, generated by mapping reads to the Typhi CT18 reference genome (AL513382)
Note the SNP coordinates used here for genotyping are relative to Typhi CT18 (AL513382)
so the input MUST be a BAM obtained via mapping to this reference sequence.
--ref Reference sequence file used for mapping (fasta).
--ref_id Name of the Typhi CT18 chromosome reference in your VCF file.
This is the entry in the first column (#CHROM) of the data part of the file.
This is necessary in case you have mapped to multiple replicons (e.g. chromosome and
plasmid) and all the results appear in the same VCF file.
--samtools_location Specify the location of the folder containing the SAMtools installation if not standard/in path [optional]
--bcftools_location Specify the location of the folder containing the bcftools installation if not standard/in path [optional]
--vcf Path to one or more VCF files, generated by mapping reads to the Typhi CT18 reference genome (AL513382)
Note the SNP coordinates used here for genotyping are relative to Typhi CT18 (AL513382)
so the input MUST be a VCF obtained via mapping to this reference sequence.
--vcf Path to one or more VCF files generated by mapping assemblies to the Typhi CT18 reference genome (AL513382)
using ParSNP (--ref_id is optional, default value is '1').
--phred Minimum phred quality to count a variant call vs CT18 as a true SNP (default 20)
--min_prop Minimum proportion of reads required to call a SNP (default 0.1)
--output Specify the location of the output text file (default genotypes_[timestamp].txt)
Output is to standard out, in tab delimited format.
The script works by looking for SNPs that define nested levels of phylogenetic lineages for S. Typhi: primary clusters, clades and subclades. These are named in the form 1.2.3, which indicates primary cluster 1, clade 1.2, and subclade 1.2.3. For H58 the script will call lineages 1 and 2 as 4.3.1.1 and 4.3.1.2.
All genotypes implicated by the detected SNPs will be reported (comma separated if multiple are found).
Usually subclade, clade and primary cluster SNPs will be compatible, i.e. you would expect to see 1.2.3 in the subclade column, 1.2 in the clade column and 1 in the primary clade column.
The first column 'Final_call' indicates the highest resolution genotype (subclade > clade > primary clade) that could be called.
As recombination is extremely rare in S. Typhi, it is unlikely that DNA isolated from a single S. Typhi culture would have high quality SNPs that are compatible with multiple genotypes, or incompatible clade/subclade combinations... therefore if you see this in the output, it is worth investigating further whether you have contaminated sequence data or a genuine recombination. To provide very basic assistance with this the output file indicates, for each genotype-defining SNP detected, the proportion of reads that support the genotype call. A genuine recombinant strain would have p=1 for multiple incompatible SNPs, whereas a mixed DNA sample might have p~0.5. Note that if you are genotyping from assemblies, we have no information on read support to work with; instead you will see an 'A' in the Final_call_support column to indicate this result comes from a genome assembly.
WARNING: Note the reference genome CT18 has the genotype 3.2.1. It is therefore possible that unexpected behaviour (e.g. wrong reference, unspecified reference name, or problems that results in overall poor quality SNP calls in the VCF) can sometimes result in calls of 3.2.1 ... so if you see lots of strains being assigned this genotype, it is worth investigating further.
We recommend using Bowtie2 to align reads to the CT18 reference, and SAMtools to convert the *.sam file to the *.bam format. The resulting bam file(s) can be passed directly to this script via --bam. Please note the differences in the commands listed below when using SAMtools v1.1/1.2 vs. SAMtools v1.3.1 to generate bam files.
For example:
# Download CT18 (AL513382) and unzip the CT18 reference genome
wget -O CT18.fasta.gz ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/bacteria/Salmonella_enterica/reference/GCA_000195995.1_ASM19599v1/GCA_000195995.1_ASM19599v1_genomic.fna.gz
gunzip CT18.fasta.gz
# Separate the chromosome sequence from the plasmids with the emboss toolkit
seqretsplit CT18.fasta
mv nc_003198.1.fasta CT18.fasta
# Replace the header line of the CT18.fasta file with the reference id i.e. chage
>NC_003198.1 Salmonella enterica subsp. enterica serovar Typhi str. CT18, complete genome
# to
> AL513382.1
# Download reads for S. Typhi 8(04)N, isolated from Vietnam in 2004
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR343/ERR343343/ERR343343_1.fastq.gz
wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/ERR343/ERR343343/ERR343343_2.fastq.gz
# Use bowtie to map reads to the CT18 reference genome
For example, to align paired end reads to the CT18 reference genome sequence:
bowtie2-build CT18.fasta CT18
bowtie2 -p 2 -x CT18 -1 ERR343343_1.fastq.gz -2 ERR343343_2.fastq.gz -S output.sam
samtools view -bS output.sam > unsorted_output.bam
samtools sort unsorted_output.bam output
(or, 'samtools sort unsorted_output.bam > output.bam' for SAMtools v1.3.1 instead of SAMtools v1.2/1.1)
# Call Typhi genotypes from the resulting BAM(s)
python genotyphi.py --mode bam --bam output.bam --ref CT18.fasta --ref_id AL513382.1 --output genotypes_test.txt
Expected output:
For bam output the column 'Number of SNPs called' reports the number of SNPs present in the resultant VCF file(s).
File Final_call Final_call_support Subclade Clade PrimaryClade Support_Subclade Support_Clade Support_PrimaryClade Number of SNPs called QRDR mutations
output.vcf 4.3.1.2 1.0 4.3.1.2 4 1.0 1.0 77 gyrA-D87G
We recommend using ParSNP to align genomes to the CT18 reference. The resulting multi-sample VCF file(s) can be passed directly to this script via --vcf_parsnp.
For example:
# Download CT18 reference genome
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/bacteria/Salmonella_enterica/reference/GCA_000195995.1_ASM19599v1/GCA_000195995.1_ASM19599v1_genomic.fna.gz
gunzip GCA_000195995.1_ASM19599v1_genomic.fna.gz
# Separate CT18 chromosome with emboss
seqretsplit GCA_000195995.1_ASM19599v1_genomic.fna
mv al513382.1.fasta CT18.fasta
# Download two example Typhi genomes for genotyping
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/245/535/GCA_000245535.1_ASM24553v1/GCA_000245535.1_ASM24553v1_genomic.fna.gz
wget ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/007/545/GCA_000007545.1_ASM754v1/GCA_000007545.1_ASM754v1_genomic.fna.gz
gunzip GCA_000245535.1_ASM24553v1_genomic.fna.gz
gunzip GCA_000007545.1_ASM754v1_genomic.fna.gz
mkdir genomes/
mv GCA_000245535.1_ASM24553v1_genomic.fna genomes/Pstx12.fasta
mv GCA_000007545.1_ASM754v1_genomic.fna genomes/Ty2.fasta
# Use ParSNP and Harvest tools to generate variant calls (VCF) for these genomes against the CT18 reference sequence
parsnp -r CT18.fasta -d genomes/ -o output
harvesttools -i ./output/parsnp.ggr -V parsnp.vcf
# Call Typhi genotypes from the resulting VCF
python genotyphi.py --mode vcf_parsnp --vcf parsnp.vcf --ref_id AL513382.1 --output genotypes_parsnptest.txt
Expected Output:
P-stx-12 should be called as clade 4.3.1 (otherwise known as H58), Ty2 should be called as clade 4.1.
The 'A' in the Final_call_support indicates the genotype calls were made from assembled data, hence no read support information is available.
File Final_call Final_call_support Subclade Clade PrimaryClade Support_Subclade Support_Clade Support_PrimaryClade
Pstx12.fasta 4.3.1 A 4.3.1 4
Ty2.fasta 4.1 A 4.1 4