Error running PStrain_V30.py script about pstrain

[sahilrishav2]

I have resolved the above issue but again new issue came:

python3 PStrain_V30.py --metaphlan3 /home/rishav/.local/bin/metaphlan --dbdir_V30 ../db_V30 -c ../test/config.txt -o test_dir --bowtie2 /usr/bin/bowtie2 --bowtie2-build /usr/bin/bowtie2-build --samtools /home/rishav/anaconda3/envs/biobakery3/bin/samtools --picard /home/rishav/packages/picard-tools-2.1.0/picard.jar --gatk /home/rishav/packages/GATK_3.5/GenomeAnalysisTK.jar

256979 pairs aligned 0 times concordantly or discordantly; of these:
513958 mates make up the pairs; of these:
513451 (99.90%) aligned 0 times
507 (0.10%) aligned exactly 1 time
0 (0.00%) aligned >1 times
0.34% overall alignment rate
[Fri Apr 26 16:30:01 IST 2024] picard.sam.AddOrReplaceReadGroups INPUT=test_dir/test//map/mapped.sort.bam OUTPUT=test_dir/test/map/mapped.bam RGLB=whatever RGPL=illumina RGPU=whatever RGSM=whatever RGID=1 VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=STRICT COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false GA4GH_CLIENT_SECRETS=client_secrets.json
[Fri Apr 26 16:30:01 IST 2024] Executing as rishav@mjlab-Precision-7920-Tower on Linux 6.5.0-27-generic amd64; OpenJDK 64-Bit Server VM 17.0.3-internal+0-adhoc..src; Picard version: 2.1.0(25ebc07f7fbaa7c1a4a8e6c130c88c1d10681802_1454776546) JdkDeflater
INFO 2024-04-26 16:30:01 AddOrReplaceReadGroups Created read group ID=1 PL=illumina LB=whatever SM=whatever

[Fri Apr 26 16:30:02 IST 2024] picard.sam.AddOrReplaceReadGroups done. Elapsed time: 0.00 minutes.
Runtime.totalMemory()=2181038080

ERROR ------------------------------------------------------------------------------------------

ERROR A USER ERROR has occurred (version 3.5-0-g36282e4):

ERROR

ERROR This means that one or more arguments or inputs in your command are incorrect.

ERROR The error message below tells you what is the problem.

ERROR

ERROR If the problem is an invalid argument, please check the online documentation guide

ERROR (or rerun your command with --help) to view allowable command-line arguments for this tool.

ERROR

ERROR Visit our website and forum for extensive documentation and answers to

ERROR commonly asked questions http://www.broadinstitute.org/gatk

ERROR

ERROR Please do NOT post this error to the GATK forum unless you have really tried to fix it yourself.

ERROR

ERROR MESSAGE: Invalid command line: Malformed walker argument: Could not find walker with name: HaplotypeCaller

ERROR ------------------------------------------------------------------------------------------

[E::hts_open_format] Failed to open file "test_dir/test//map/mapped.vcf.gz" : No such file or directory
No need to merge in case of one sample.

[wshuai294]

Hello, how did you resolve the first issue? It seems that no read is mapped to the reference.

[sahilrishav2]

Actually, the previous problem was coming from bowtie2 . Bowtie2 was not properly installed

[sahilrishav2]

This problem is however from GATK. I am unable to solve it. Kindly help me

[wshuai294]

Is the file test_dir/test//ref//merged_ref.fa empty? How about the content in the metaphlan3 output and the bam file? I guess the problem is not caused by GATK. Because no reads are mapped successfully, seen from

256979 pairs aligned 0 times concordantly or discordantly; of these:
513958 mates make up the pairs; of these:
513451 (99.90%) aligned 0 times
507 (0.10%) aligned exactly 1 time
0 (0.00%) aligned >1 times
0.34% overall alignment rate

[wshuai294]

I am more than happy to help you with this issue. Now I am trying to reproduce this error.

[sahilrishav2]

Yaa, now, I get it. Currently, i am just running the test datasets provided by you as a checking process. So, i want to ask if the command that i am running is working well ? could i use this on my real data

[wshuai294]

I guess there is some error. Your metaphlan3 output might be empty. I guess there might be a problem with your metaphlan. Is the version of /home/rishav/.local/bin/metaphlan methphlan3?

[sahilrishav2]

One more thing i want to say that i am using metaphlan4 and metaphlan4 database

[wshuai294]

there might be a problem because, in this case, the metaphlan output is inconsistent with --dbdir_V30 ../db_V30.

[sahilrishav2]

So, isn't there a way to use it with metaphlan4

[wshuai294]

Currently, it cannot. But if you need this, I will try to update it to support metaphlan4.

[sahilrishav2]

Actually, i am performing benchmarking of some tools in which i have included PStrain also. So, it would be better if you upgrade it to support MetaPhlan4, so that i could use this tool in its best way.

[wshuai294]

Ok, Great to hear this. I will upgrade it soon. Also, I will prepare a conda env to make it easier to install.

[sahilrishav2]

Thank you so much for your support

[wshuai294]

Hello, I am glad to inform you that PStrain supports Metaphlan 4 now. I also provided the conda environment to easily use it. Please follow this:

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 pstrain

Then get the metaphlan database. If you have the database already, you can skip this step, and pass the database folder and index to PStrain.py by --bowtie2db and -x (same as the Metaphlan parameter).

bash scripts/collect_metaphlan_datbase.sh -x mpa_vOct22_CHOCOPhlAnSGB_202403 -m 4 -d ./   #constructing the database for Metaphlan4 

Finally, test PStrain by:

python3 scripts/PStrain.py --help
cd test/
bash test_PStrain_V40.sh  # use Metaphlan 4, remember edit the --bowtie2db and -x if you have downloaded the database yourself.

Thank you so much for benchmarking our method. Please feel free to ask me if you have any further queries.

[sahilrishav2]

Thank you so much for your support. We will inform you whenever our paper goes in the communication process.

[wshuai294]

Hello, I have fixed a bug related to missing NCBI_ID in the Metaphlan results. This bug was causing the process to be terminated during execution. Please pull the latest commit to get the fix. Apologies for any inconvenience caused.

[sahilrishav2]

Ok, thank you. Actually, I have not run the tool yet. Currently, i am doing standardization of some other tools also. I will get back to you if any issues persist. Thank you once again