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SpatialTranscriptomicsResearch / st_pipeline

ST Pipeline contains the tools and scripts needed to process and analyze the raw files generated with the Spatial Transcriptomics method in FASTQ format.
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Error STAR #111

Closed alessandriLuca closed 4 years ago

alessandriLuca commented 5 years ago

Good morning, first of all i would like to congrats for the pipeline and the work done! I think that this is very useful! Im trying to run the test but after the script load successfully all the data then i receive this error :

log_file.txt

This is the way in which im calling the function : python3 st_pipeline_run.py --expName test --ids /data2/st_pipeline-master/ids/1000L2_barcodes.txt --ref-map /data2/st_pipeline-master/tests/config/contaminant_genomes/R45S5_R5S1/ --log-file log_file.txt --output-folder /home/me/results --ref-annotation /data2/st_pipeline-master/tests/config/annotations/Homo_sapiens.GRCh38.79_chr19.gtf /data2/st_pipeline-master/tests/input/arrayjet_1002/testdata_R1.fastq /data2/st_pipeline-master/tests/input/arrayjet_1002/testdata_R2.fastq and im using all the data that are in your folders.

Thanks in advance for your availability.

Best

Luca

jfnavarro commented 5 years ago

Hi Luca!

Thanks for your message.

You need to have the aligner STAR installed in your system.

https://github.com/alexdobin/STAR

alessandriLuca commented 5 years ago

Hi! Thats was a super fast answer! Thanks gonna install and try!

alessandriLuca commented 5 years ago

Hi Josè, so i just fixed STAR but i have another problem, 

ST Pipeline, parameters loaded
ST Pipeline, logger created
ST Pipeline, sanity check passed. Starting the run.
Error running the pipeline
Error mapping with STAR.
Output file not present /tmp/st_pipeline_temp4_tikme4/Aligned.sortedByCoord.out.bam
b'/tmp/st_pipeline_temp4_tikme4/_STARtmp//readsCommand_read1: 3: /tmp/st_pipeline_temp4_tikme4/_STARtmp//readsCommand_read1: samtools: not found\nsh: 1: samtools: not found\n\nEXITING because of FATAL ERROR: could not open genome file /data2/st_pipeline-master/tests/config/contaminant_genomes/R45S5_R5S1/genomeParameters.txt\nSOLUTION: check that the path to genome files, specified in --genomeDir is correct and the files are present, and have user read permsissions\n\nAug 08 13:18:23 ...... FATAL ERROR, exiting\n'

Maybe im using in the wrong way the example data that you have in github?

jfnavarro commented 5 years ago

As it says in the error message you also need to install samtools which is a requirement for STAR to work...

alessandriLuca commented 5 years ago

Hi Josè, as you can see from the picture samtools is already installed. image

Thank you again

alessandriLuca commented 5 years ago

Dear Josè, i tried several fix but i got still the same error, do you have some ideas?

jfnavarro commented 5 years ago

Are you able to run STAR and map some test file? What STAR version are you using?

alessandriLuca commented 5 years ago

Dear Josè, i reinstalled everything in order to be sure to have the right versions that you suggest in your tutorial. Right now with the same code python3 st_pipeline_run.py --expName test --ids /data2/st_pipeline-master/ids/1000L2_barcodes.txt --ref-map /data2/st_pipeline-master/tests/config/contaminant_genomes/R45S5_R5S1/ --log-file log_file.txt --output-folder /home/me/results --ref-annotation /data2/st_pipeline-master/tests/config/annotations/Homo_sapiens.GRCh38.79_chr19.gtf /data2/st_pipeline-master/tests/input/arrayjet_1002/testdata_R1.fastq /data2/st_pipeline-master/tests/input/arrayjet_1002/testdata_R2.fastq

i have another error: Output file not present /tmp/st_pipeline_tempciaq7pap/Aligned.sortedByCoord.out.bam b'\nEXITING because of FATAL ERROR: could not open genome file /data2/st_pipeline-master/tests/config/contaminant_genomes/R45S5_R5S1/genomeParameters.txt\nSOLUTION: check that the path to genome files, specified in --genomeDir is correct and the files are present, and have user read permsissions\n\nAug 14 09:48:20 ...... FATAL ERROR, exiting\n'

samtools and Star works Perfectly. Seems that i miss something in the genome directory but im using your example files. Thank you again

Best

Luca

jfnavarro commented 5 years ago

Ahhhh, I know what is going on. You need to create the genome indexes. I provide the .fasta files but you need to use STAR to create the genome indexes for the genome and the contaminant genome. The indexes will be composed of several files and folders and you need to pass the pipeline the path to the index's folder. Read the STAR documentation to know more about how to create indexes. It is straight forward. If you read the documentation it says that you need to pass a genome index not a fasta file as argument, I should be more clear with this in the documentation :)

alessandriLuca commented 5 years ago

Oh ok! Yes i can create the genome index, i thought that would have been created from the script itself! Thank you ;)

jfnavarro commented 5 years ago

Oh no, you need to create the index only once. It would not be smart to let the pipeline create the index every time you run it. Okay, let me know if you manage to make it work so I can close the issue.

alessandriLuca commented 5 years ago

Yes you are right! Ok cool i will let you know ;) Thanks again!

alessandriLuca commented 5 years ago

Perfect is working!! Thank you again!