First, get the source code and construct the env
git clone https://github.com/wshuai294/PStrain.git --depth 1
cd PStrain/
conda env create --name pstrain -f pstrain_metaphlan4_env.yml
conda activate pstrainThen get the metaphlan database
bash scripts/collect_metaphlan_datbase.sh -x mpa_vOct22_CHOCOPhlAnSGB_202403 -m 4 -d ./ #constructing the reference for Metaphlan4
python3 scripts/PStrain.py --helptest PStrain by:
cd test/
bash test_PStrain_V40.sh # remember edit the --bowtie2db and -x if you have downloaded the database yourself.Also, you can install it using Bioconda directly by:
conda create --name pstrain -c bioconda -c conda-forge pstrain
conda activate pstrainor install it in an existing environment:
conda install bioconda::pstrainFirst, get the source code and construct the env
git clone https://github.com/wshuai294/PStrain.git --depth 1
cd PStrain/
conda env create --name pstrain3 -f pstrain_metaphlan3_env.yml
conda activate pstrain3Then get the metaphlan database
bash scripts/collect_metaphlan_datbase.sh -x mpa_v31_CHOCOPhlAn_201901 -m 3 -d ./ #constructing the reference for Metaphlan3
python3 scripts/PStrain.py --helptest PStrain by:
cd test/
bash test_PStrain_V30.sh # remember edit the --bowtie2db and -x if you have downloaded the database yourself.git clone https://github.com/wshuai294/PStrain.git --depth 1
cd PStrain/
conda env create --name pstrain2 -f pstrain_metaphlan2_env.yml
conda activate pstrain2
python3 scripts/PStrain_m2.py -hDownload db/ and packages/ from Google Drive or Zenodo, and put them in the same path with the scripts/ folder. Also, you can appoint the path of the dependencies by --dbdir, --prior and --metaphlan2.
test PStrain (with Metaphlan2) by:
cd test/
bash test_run.shNote:
| Scripts | Description |
|---|---|
| scripts/PStrain.py | Strain profiling based on the Metaphlan4 or Metaphlan3 |
| scripts/PStrain_m2.py | Strain profiling based on the Metaphlan2 |
| scripts/collect_metaphlan_datbase.sh | collect the Metaphlan4/3 database and build the bowtie2 index |
| scripts/single_species.py | phase variants for a single genome, based on the BAM and VCF file |
| scripts/merge.py | Merge the strain results for downstream analyses |
usage: python3 PStrain.py [options] -c/--conf <config file> -o/--outdir <output directory> --bowtie2db <metaphlan database> -x <metaphlan db index>
Example: python3 PStrain.py -c config.txt -o out --bowtie2db ../mpa_vOct22_CHOCOPhlAnSGB_202403/ -x mpa_vOct22_CHOCOPhlAnSGB_202403 # Metaphlan 4
python3 PStrain.py --metaphlan_version 3 -c config.txt -o outdir/ --bowtie2db ../mpa_v31_CHOCOPhlAn_201901/ -x mpa_v31_CHOCOPhlAn_201901 # Metaphlan 3
The config file should follow the format:
//
sample: [sample1_ID]
fq1: [forward reads fastq]
fq2: [reverse/mate reads fastq]
//
sample: [sample2_ID]
fq1: [forward reads fastq]
fq2: [reverse/mate reads fastq]
...
Help information can be found by python3 PStrain.py -h/--help, config file format for single end reads , and additional information can be found in README.MD or https://github.com/wshuai294/PStrain.
PStrain: profile strains in shotgun metagenomic sequencing reads.
required arguments:
-c , --conf The configuration file of the input samples. (default: None)
-o , --outdir The output directory. (default: None)
--bowtie2db Path to metaphlan bowtie2db. (default:
/home/wangshuai/softwares/PStrain/scripts/../mpa_vOct22_CHOCOPhlAnSGB_202403)
-x , --metaphlan_index
metaphlan bowtie2db index. (default: mpa_vOct22_CHOCOPhlAnSGB_202403)
options:
-h, --help show this help message and exit
--metaphlan_version Metaphlan version [3 or 4]. Used to download the corresponding database. If you build the
metaphlan database yourself, just ignore this. (default: 4)
--bowtie2 Path to bowtie2 binary. (default: bowtie2)
--bowtie2-build Path to bowtie2 binary. (default: bowtie2-build)
--samtools Path to samtools binary. (default: samtools)
--metaphlan Path to metaphlan. (default: metaphlan)
--metaphlan_output_files
If you have run metaphlan already, give metaphlan result file in each line, the order should be
the same with config file. In particular, '--tax_lev s' should be added while running
metaphlan. (default: )
-p , --proc The number of process to use for parallelizing the whole pipeline, run a sample in each
process. (default: 1)
-n , --nproc The number of CPUs to use for parallelizing the mapping with bowtie2. (default: 1)
-w , --weight The weight of genotype frequencies while computing loss, then the weight of linked read type
frequencies is 1-w. The value is between 0~1. (default: 0.3)
--lambda1 The weight of prior knowledge while rectifying genotype frequencies. The value is between 0~1.
(default: 0.0)
--lambda2 The weight of prior estimation while rectifying second order genotype frequencies. The value is
between 0~1. (default: 0.0)
--species_dp The minimum depth of species to be considered in strain profiling step (default is 5).
(default: 5)
--snp_ratio The SNVs of which the depth are less than the specific ratio of the species's mean depth would
be removed. (default: 0.45)
--qual The minimum quality score of SNVs to be considered in strain profiling step. (default: 20)
--similarity The similarity cutoff of hierachical clustering in merge step. (default: 0.95)
--elbow The cutoff of elbow method while identifying strains number. If the loss reduction ratio is
less than the cutoff, then the strains number is determined. (default: 0.24)
--gatk Path to gatk binary. (default: /home/wangshuai/softwares/PStrain/scripts//GenomeAnalysisTK.jar)
--picard Path to picard binary. (default: /home/wangshuai/softwares/PStrain/scripts//picard.jar)
--prior The file of prior knowledge of genotype frequencies in the population. Not providing this is
also ok. (default: None)If you have run metaphlan3 already for each file, to save runtime, you can build a file to record
the metaphlan3 resultfile of each sample in each line (In particular, --tax_lev s should be added while running metaphlan3.).
Please afford this file to PStrain.py with the parameter --metaphlan_output_files.
The file should be like
sample1_metaphlan_output.txt
sample2_metaphlan_output.txt
sample3_metaphlan_output.txt
...Then PStrain will skip running metaphlan, and use the existing metaphlan result files.
python3 PStrain.py --metaphlan_output_files exisiting_metaphlan_results.txt -c test.config -o test_dir For metaphlan3/4, we have not built prior genotype frequencies database which will not impact the performance very much. So just ignore --prior parameter. Also, you can build a prior genotype frequencies database by yourself. I will show how to do this in the following section. In this way, PStrain will run Metaphlan to find the species profile in the sample and profile the strains in each species.
Example:
Example: python3 PStrain_m2.py -p 10 -n 8 --similarity 0.95 -o ./outdir -c config.txt \
--dbdir /home/wangshuai/strain/00.simulation/15.needle/db \
--prior /home/wangshuai/strain/00.simulation/15.needle/db/prior_beta.pickle\
--metaphlan2 /path-to/metaphlan2Command:
PStrain: profile strains in shotgun metagenomic sequencing reads.
required arguments:
-c , --conf The configuration file of the input samples. (default:
None)
-o , --outdir The output directory. (default: None)
--dbdir Path to marker gene database, can be downloaded from
https://zenodo.org/doi/10.5281/zenodo.10457543. (default:
/home/wangshuai/softwares/PStrain/scripts/../db/)
optional arguments:
-h, --help show this help message and exit
-p , --proc The number of process to use for parallelizing the whole
pipeline, run a sample in each process. (default: 1)
-n , --nproc The number of CPUs to use for parallelizing the mapping
with bowtie2. (default: 1)
-w , --weight The weight of genotype frequencies while computing loss,
then the weight of linked read type frequencies is 1-w.
The value is between 0~1. (default: 0.3)
--lambda1 The weight of prior knowledge while rectifying genotype
frequencies. The value is between 0~1. (default: 0.0)
--lambda2 The weight of prior estimation while rectifying second
order genotype frequencies. The value is between 0~1.
(default: 0.0)
--species_dp The minimum depth of species to be considered in strain
profiling step. (default: 5)
--snp_ratio The SNVs of which the depth are less than the specific
ratio of the species's mean depth would be removed.
(default: 0.45)
--qual The minimum quality score of SNVs to be considered in
strain profiling step. (default: 20)
--similarity The similarity cutoff of hierachical clustering in merge
step. (default: 0.95)
--elbow The cutoff of elbow method while identifying strains
number. If the loss reduction ratio is less than the
cutoff, then the strains number is determined. (default:
0.24)
--bowtie2 Path to bowtie2 binary. (default: bowtie2)
--bowtie2-build Path to bowtie2 binary. (default: bowtie2-build)
--samtools Path to samtools binary. (default: samtools)
--metaphlan Path to metaphlan. (default: /home/wangshuai/softwares/PSt
rain/scripts/../packages/metaphlan2/metaphlan2.py)
--gatk Path to gatk binary. (default: /home/wangshuai/softwares/P
Strain/scripts//GenomeAnalysisTK.jar)
--picard Path to picard binary. (default:
/home/wangshuai/softwares/PStrain/scripts//picard.jar)
--prior The file of prior knowledge of genotype frequencies in the
population. (default: /home/wangshuai/softwares/PStrain/sc
ripts/../db/prior_beta.pickle)
--skip_prior Whether use prior knowledge of genotype frequencies.
(default: False)To use single-end data, the only difference is in the config file, just leave the fq2: line empty. An config example for single-end data is:
//
sample: [sample1_ID]
fq1: [single-end reads fastq]
fq2:
//
sample: [sample2_ID]
fq1: [single-end reads fastq]
fq2: In the output directory specified by -o, there are directories for each sample named by sample ID and one directory named merge.
In the directory of one specific sample, you will find a folder called result, in which you can find the below files and directories:
strain_RA.txt
strain_merged_RA.txt
species1_seq.txt
species2_seq.txt
...
The formats for strain_RA.txt and strain_merged_RA.txt are similar. An example is as below:
# Species Species_RA Strain_ID Cluster_ID Strain_RA
Escherichia_coli 88.00 str-1 clu-1 0.1
Escherichia_coli 88.00 str-2 clu-2 0.2
Escherichia_coli 88.00 str-3 clu-3 0.7
Leifsonia_rubra 12.00 str-1 clu-1 0.4
Leifsonia_rubra 12.00 str-2 clu-2 0.6This means in this sample, there are two species: Escherichia coli with three strains and Leifsonia rubra with two strains.
Species_RA column means the species relative abundance in the sample.
Strain_ID column means the strains ID of each species in the sample.
Cluster_ID column means the strains' corresponding cluster in all of the samples.
Strain_RA column means the strains abundance of each species in the sample.
In the merge directory, you can find below files.
strain_number.txt
species_1_seq.txt
species_1_clu.txt
species_2_seq.txt
species_2_clu.txt
...strain_number.txt contains the strain number of each species in all of the samples.
species_seq.txt contains the strain sequence of in all of the samples.
species_clu.txt contains the consensus sequence of each strains cluster in all of the samples.
The file listed below is one *_clu.txt example, the formate of *_clu.txt and *_seq.txt are similar.
# Gene Locus Ref Alt clu-1 clu-2 clu-3 clu-4 clu-5
gi|545276179|ref|NZ_KE701962.1|:c548151-546829 90 T G 1 0 0 1 1
gi|545276179|ref|NZ_KE701962.1|:c548151-546829 303 G A 0 1 0 1 1
gi|545276179|ref|NZ_KE701962.1|:c548151-546829 310 A T 0 0 1 0 1
gi|545276179|ref|NZ_KE701962.1|:c548151-546829 312 C A 1 1 1 0 0
...Gene, Locus, Ref, and Alt columns represent the locus, following columns mean the composition of the consensus sequence of each cluster.
First, we can use merge.py to cluster the strains obtained from all the samples with user-defined parameters.
usage: python3 merge.py [options] -c/--conf <config file> -o/--outdir <output directory>
Merge the results from all of the samples.
required arguments:
-c , --conf The configuration file of the input samples, which is same as mentioned above.
-o , --outdir The output directory.
optional arguments:
-h, --help show this help message and exit
--similarity The similarity cutoff of hierachical clustering in merge step (default is 0.95).Furthermore, we can adopt PStrain-tracer for downstream analysis and visualization.
Please refer to PStrain-tracer.
If there is only one species, you can map reads and call SNPs by yourself. After that, you can use single_species.py from scripts/ folder to profile strains.
If you have the prior knowledge of strains number, you can add this info to argument -k/--strainsNum. Once the -k/--strainsNum argument is set to 1, the consensus sequence with major alleles will be generated.
Detailed usage information can be found as follows:
Usage: python3 single_species.py [options] -b/--bam <bam file> -v/--vcf <vcf file> -o/--outdir <output directory>
Example: python3 single_species.py -b mapped.bam -v mapped.vcf.gz --prior /home/wangshuai/strain/00.simulation/15.needle/db/prior_beta.pickle -o phase_result -k 2
Before using this script, you should align the reads to your reference, and call SNPs. Then you can provide the bam and vcf file. Once you have the prior knowledge of the strains number, you can provie it the to script by -k/--strainsNum parameter. Otherwise,it will choose the strains number by elbow method.
Help information can be found by python3 single_species.py -h/--help, additional information can be found in README.MD or https://github.com/wshuai294/PStrain.
PStrain: phase strains in samples with single species.
required arguments:
-b , --bam The bam file of the input samples.
-v , --vcf The vcf file of the input samples.
-o , --outdir The output directory.
optional arguments:
-h, --help show this help message and exit
-k , --strainsNum The number of strains in the sample (default is chosen
by elbow method).
--snp_dp The minimum depth of SNPs to be considered in strain
profiling step (default is 5).
--prior The file of prior knowledge of genotype frequencies in
the population.
--elbow The cutoff of elbow method while identifying strains
number. If the loss reduction ratio is less than the
cutoff, then the strains number is determined.
--qual The minimum quality score of SNVs to be considered in
strain profiling step (default is 20).
-w , --weight The weight of genotype frequencies while computing loss,
then the weight of linked read type frequencies is 1-w.
The value is between 0~1. (default is 0.0)
--lambda1 The weight of prior knowledge while rectifying genotype
frequencies. The value is between 0~1. (default is 0.0)
--lambda2 The weight of prior estimation while rectifying second
order genotype frequencies. The value is between 0~1.
(default is 0.0)You can build the species-specific pipeline easily by yourself, the code single_species.py in the scripts/ folder can be very useful.
You can build the pipeline as follow:
Step 1. Given the raw fastq file of a sample, run Metaphlan3 to get the species
abundance.
Step 2. For each species in the sample, extract the species' corresponding marker
genes from Metaphlan3 as the reference.
Step 3. For each species, map sample's reads to the reference of the species with
bowtie2 and call SNV with GATK, you will get the BAM and VCF file.
Step 4. For each species, run python3 single_species.py [options] -b/--bam <bam file>
-v/--vcf <vcf file> -o/--outdir <output directory>.
Step 5. Then you get the strain profiles of each species in each sample, you can now
analyze them. You can also use other marker gene database in the same way.
Although PStrain's performance is good enough without the prior database except for the gene with too low depth. You can build your own prior database to improve the performance. PStrain will assume the prior beta is 0.5 if not finding prior knowledge.
To generate the prior database, please follow these steps:
locus A T C G
gi|378760316|ref|NZ_AGDG01000038.1|:8694-9629|Bacteroides_faecis_517 1 0 0 0
gi|410936685|ref|NZ_AJRF02000012.1|:54814-55320|Enterococcus_faecium_299 0 1 0 0
gi|139439993|ref|NZ_AAVN02000016.1|:7311-8633|Collinsella_aerofaciens_783 0.0102827763496144 0.0205655526992288 0 0.969151670951157
gi|239633764|ref|NZ_GG670286.1|:165060-166319|Enterococcus_gallinarum_626 0 0 0 1
gi|345651640|ref|NZ_JH114383.1|:276898-279936|Bacteroides_vulgatus_2725 0 0 0 1
GeneID:16385027|Lactococcus_phage_jm2_95 1 0 0 0
gi|488673303|ref|NZ_KB850950.1|:c879018-878179|Clostridium_hathewayi_511 0 0 0 1import pickle
popu={}
f=open('prior_beta.txt','r')
for line in f:
break
for line in f:
line=line.strip()
array=line.split()
atcg=array[1:]
popu[array[0]]=atcg
with open('prior_beta.pickle', 'wb') as f:
pickle.dump(popu, f, pickle.HIGHEST_PROTOCOL) Then please afford the file to PStrain with parameter --prior.
PStrain needs two database files, one is the metaphlan2 marker gene database, and the other is the prior genotype frequencies database. We have uploaded the two database in the db/ folder to the Google Drive, you can directly download the db/ folder, and put it in the same path with the scripts/ folder. PStrain could use them by default. Also, you can appoint the path of these two databases by corresponding arguments --dbdir and --prior.
PStrain relies on these software packages. PStrain could use them by default. Also, you can install them by yourself, and appoint them by --samtools, --bowtie2-build, --bowtie2, --metaphlan2, --gatk, and --picard arguments.
Note:
Shuai Wang, Yiqi Jiang, Shuaicheng Li, PStrain: An Iterative Microbial Strains Profiling Algorithm for Shotgun Metagenomic Sequencing Data, Bioinformatics, , btaa1056, https://doi.org/10.1093/bioinformatics/btaa1056
Should you have any queries, please feel free to contact me, I will reply as soon as possible (swang66-c@my.cityu.edu.hk).