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williamritchie / IRFinder

Detecting intron retention from RNA-Seq experiments
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Mapability directory not complete #97

Closed gabee-chan closed 4 years ago

gabee-chan commented 4 years ago

Hi ! I'm trying to built and index for Squirrel Monkey and when I run it the Mapability directory is incomplete as I only have these files:

Log.out
Log.progress.out
Log.std.out
MapabilityExclusion.bed.gz
_STARtmp

Looking at the Log.out file I get : Completed: thread #21

The first time I tried to ran it I was building the index using the GTF url but I was having an error that there were no "exon" in the transcripts.gtf file. So then I downloaded the genome and GTF file and used the command:

IRFinder -m BuildRefProcess -r $REF -e $Path_extras/RNA.SpikeIn.ERCC.fasta.gz

Where REF is the path where the genome.fa and transcripts.gtf file are (with those names)

In any of the log files I'm being able to find which could be the possible reason why the index is not being able to run. I would appreciate if you could help me.

Cheers, Gaby

dg520 commented 4 years ago

Hi @gabee-chan ,

Sorry for a late reply. I need more details about the error. To be exact, I need your full standard output and standard error messages from the beginning to the end. Meanwhile, please also provide your OS information and versions of STAR, Bedtools and Samtools. You can either post here or send me through by email.

Best, Dadi

gabee-chan commented 4 years ago

Hi Dadi,

Thanks for your help. I attached the standard output and standard error from both Squirrel Monkey and Marmoset

OS: uname -or 2.6.32-754.17.1.el6.x86_64 GNU/Linux

STAR 2.6 Bedtools v2.25.0 Samtools 1.31

Cheers, Gaby

El lun., 22 jun. 2020 a las 10:13, Dadi (notifications@github.com) escribió:

Hi @gabee-chan https://github.com/gabee-chan ,

Sorry for a late reply. I need more details about the error. To be exact, I need your full standard output and standard error messages from the beginning to the end. Meanwhile, please also provide your OS information and versions of STAR, Bedtools and Samtools. You can either post here or send me through by email.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/williamritchie/IRFinder/issues/97#issuecomment-647200839, or unsubscribe https://github.com/notifications/unsubscribe-auth/ADRQSTKZCEH4PQCZPKDAU7TRX2O25ANCNFSM4OBO27RQ .

dg520 commented 4 years ago

Hi @gabee-chan ,

I got your package versions. But I cannot find standard output and error attachments.

Best, Dadi

gabee-chan commented 4 years ago

Hi Dadi,

I re-attached the files changing the name with the suffix txt in case that was the problem. Hope that works 😓

Thanks, Gaby

El lun., 22 jun. 2020 a las 12:11, Dadi (notifications@github.com) escribió:

Hi @gabee-chan https://github.com/gabee-chan ,

I got your package versions. But I cannot find standard output and error attachments.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/williamritchie/IRFinder/issues/97#issuecomment-647227317, or unsubscribe https://github.com/notifications/unsubscribe-auth/ADRQSTOEBR74F5MYJAHZWYTRX24VFANCNFSM4OBO27RQ .

Warning: no access to tty (Bad file descriptor). Thus no job control in this shell. Launching reference build process. The full build should take at least one hour. Usage : /share/ClusterShare/biodata/contrib/gabrod/anaconda2/opt/irfinder-1.2.5/bin/util/IRFinder-BuildRefFromEnsembl mode threads STAR-executable base_ftp_url_of_ensembl_genome+gtf output_directory(must not exist) additional_genome_reference(eg: ERCC) non_polyA_genes-as-bed region_blacklist-as-bed Usage example: /share/ClusterShare/biodata/contrib/gabrod/anaconda2/opt/irfinder-1.2.5/bin/util/IRFinder-BuildRefFromEnsembl BuildRef 12 STAR "ftp://ftp.ensembl.org/pub/release-75/fasta/homo_sapiens/dna/" "IRFinder/REF/Human" "Refernce-ERCC.fa.gz" [non_polyA_genes.bed] [blacklist.bed] Trying to fetch dna.primary_assembly and GTF based on: ftp://ftp.ensembl.org/pub/release-99/gtf/callithrix_jacchus/Callithrix_jacchus.ASM275486v1.99.gtf.gz

Jun 15 16:08:41 ..... started STAR run Jun 15 16:08:41 ... starting to generate Genome files Jun 15 16:11:17 ... starting to sort Suffix Array. This may take a long time... Jun 15 16:12:36 ... sorting Suffix Array chunks and saving them to disk... Jun 15 16:38:05 ... loading chunks from disk, packing SA... Jun 15 16:39:47 ... finished generating suffix array Jun 15 16:39:47 ... generating Suffix Array index Jun 15 16:44:50 ... completed Suffix Array index Jun 15 16:44:50 ..... processing annotations GTF Jun 15 16:45:02 ..... inserting junctions into the genome indices Jun 15 16:48:34 ... writing Genome to disk ... Jun 15 16:49:32 ... writing Suffix Array to disk ... Jun 15 16:51:27 ... writing SAindex to disk Jun 15 16:51:34 ..... finished successfully Star genome build result: 0 Commence STAR mapping run for mapability. Mon Jun 15 16:51:34 AEST 2020 Completed STAR run. Mon Jun 15 18:07:58 AEST 2020 Commence Coverage calculation. Completed coverage exclusion calculation. Mon Jun 15 18:41:54 AEST 2020 Mapability result: 0 Build Ref 1 Build Ref 2 Build Ref 3 Build Ref 4 Build Ref 5 Build Ref 6 Build Ref 7 Build Ref 8 Build Ref 9 Build Ref 10 Build Ref 11 Build Ref 12 Build Ref 13c Build Ref 14c Build Ref 16 - COMPLETE Ref build result: 0 ALL DONE

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real 44m1.549s user 316m4.000s sys 18m27.368s

real 38m54.818s user 970m46.571s sys 268m51.205s

real 0m2.140s user 0m2.007s sys 0m0.138s

Warning: no access to tty (Bad file descriptor). Thus no job control in this shell. Launching reference build process. The full build should take at least one hour. Usage : /share/ClusterShare/biodata/contrib/gabrod/anaconda2/opt/irfinder-1.2.5/bin/util/IRFinder-BuildRefFromEnsembl mode threads STAR-executable base_ftp_url_of_ensembl_genome+gtf output_directory(must not exist) additional_genome_reference(eg: ERCC) non_polyA_genes-as-bed region_blacklist-as-bed Usage example: /share/ClusterShare/biodata/contrib/gabrod/anaconda2/opt/irfinder-1.2.5/bin/util/IRFinder-BuildRefFromEnsembl BuildRef 12 STAR "ftp://ftp.ensembl.org/pub/release-75/fasta/homo_sapiens/dna/" "IRFinder/REF/Human" "Refernce-ERCC.fa.gz" [non_polyA_genes.bed] [blacklist.bed] Jun 18 16:33:33 ..... started STAR run Jun 18 16:33:33 ... starting to generate Genome files Jun 18 16:34:56 ... starting to sort Suffix Array. This may take a long time... Jun 18 16:35:36 ... sorting Suffix Array chunks and saving them to disk... Jun 18 16:50:31 ... loading chunks from disk, packing SA... Jun 18 16:51:53 ... finished generating suffix array Jun 18 16:51:53 ... generating Suffix Array index Jun 18 16:56:44 ... completed Suffix Array index Jun 18 16:56:44 ..... processing annotations GTF Jun 18 16:56:56 ..... inserting junctions into the genome indices Jun 18 17:00:17 ... writing Genome to disk ... Jun 18 17:00:32 ... writing Suffix Array to disk ... Jun 18 17:02:21 ... writing SAindex to disk Jun 18 17:02:28 ..... finished successfully Star genome build result: 0 Commence STAR mapping run for mapability. Thu Jun 18 17:02:29 AEST 2020 Completed STAR run. Thu Jun 18 17:46:30 AEST 2020 Commence Coverage calculation. Completed coverage exclusion calculation. Thu Jun 18 18:25:29 AEST 2020 Mapability result: 0 Build Ref 1 Build Ref 2 Build Ref 3 Build Ref 4 Build Ref 5 Build Ref 6 Build Ref 7 Build Ref 8 Build Ref 9 Build Ref 10 Build Ref 11 Build Ref 12 Build Ref 13c Build Ref 14c Build Ref 16 - COMPLETE Ref build result: 0 ALL DONE

--2020-06-15 16:05:39-- ftp://ftp.ensembl.org/pub/release-99/fasta/callithrix_jacchus/dna/*.dna.primary_assembly.fa.gz => `.listing' Resolving ftp.ensembl.org... 193.62.197.76 Connecting to ftp.ensembl.org|193.62.197.76|:21... connected. Logging in as anonymous ... Logged in! ==> SYST ... done. ==> PWD ... done. ==> TYPE I ... done. ==> CWD (1) /pub/release-99/fasta/callithrix_jacchus/dna ... done. ==> PASV ... done. ==> LIST ... done.

 0K                                                        31.3K=0.03s

2020-06-15 16:05:43 (31.3 KB/s) - `.listing' saved [928]

Removed .listing'. No matches on pattern.dna.primary_assembly.fa.gz'. --2020-06-15 16:05:43-- ftp://ftp.ensembl.org/pub/release-99/fasta/callithrix_jacchus/dna/.dna.toplevel.fa.gz => `.listing' Resolving ftp.ensembl.org... 193.62.197.76 Connecting to ftp.ensembl.org|193.62.197.76|:21... connected. Logging in as anonymous ... Logged in! ==> SYST ... done. ==> PWD ... done. ==> TYPE I ... done. ==> CWD (1) /pub/release-99/fasta/callithrix_jacchus/dna ... done. ==> PASV ... done. ==> LIST ... done.

 0K                                                        66.7M=0s

2020-06-15 16:05:46 (66.7 MB/s) - `.listing' saved [928]

Removed .listing'. --2020-06-15 16:05:46-- ftp://ftp.ensembl.org/pub/release-99/fasta/callithrix_jacchus/dna/Callithrix_jacchus.ASM275486v1.dna.toplevel.fa.gz =>Callithrix_jacchus.ASM275486v1.dna.toplevel.fa.gz' ==> CWD not required. ==> PASV ... done. ==> RETR Callithrix_jacchus.ASM275486v1.dna.toplevel.fa.gz ... done. Length: 858296988 (819M)

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dg520 commented 4 years ago

Hi @gabee-chan ,

I guess these two genomes have a lot of chromosomes/contigs/scaffolds, am I right? Older versions of IRFinder are NOT able to handle genomes with tremendous contigs. But I believe I've fixed this issue in the latest version of IRFinder 1.3.0.

I also realized that you said your Samtools is version 1.31. But the latest version of Samtools is 1.10. I guess you mean 1.3.1 instead. Please note, you have to update your Samtools to at least version 1.4 before using IRFinder 1.3.0.

Best, Dadi

gabee-chan commented 4 years ago

Hi Dadi,

Thanks for your help. I re-install IRFinder and Samtools with the proper versions and was able to build the index for squirrel monkey as all the files in the IRFinder folder have an output. Nonetheless, when I tried to do the quantification step I'm getting the following irfinder.stderror:

home/gabrod/bin/IRFinder-1.3.0/bin/util/irfinder: /usr/lib64/libstdc++.so.6: version GLIBCXX_3.4.14' not found (required by /home/gabrod/bin/IRFinder-1.3.0/bin/util/irfinder) /home/gabrod/bin/IRFinder-1.3.0/bin/util/irfinder: /usr/lib64/libstdc++.so.6: versionGLIBCXX_3.4.20' not found (required by /home/gabrod/bin/IRFinder-1.3.0/bin/util/irfinder)

I looked into the /usr/lib64 directory and I have the libstdc++.so.6 -> libstdc++.so.6.0.13, but cannot find the GLIBCXX_3.4.14 and GLIBCXX_3.4.20, how can I overcome this problem?

Thanks for your help, Gaby

El mar., 23 jun. 2020 a las 0:11, Dadi (notifications@github.com) escribió:

Hi @gabee-chan https://github.com/gabee-chan ,

I guess these two genomes have a lot of chromosomes/contigs/scaffolds, am I right? Older versions of IRFinder are NOT able to handle genomes with tremendous contigs. But I believe this issue should be fixed by version 1.3.0 https://github.com/williamritchie/IRFinder.

I also realized that you said your Samtools is version 1.31. But the latest version of Samtools is 1.10. I guess you mean 1.3.1 instead. Please note, you have to update your Samtools to at least version 1.4 before using IRFinder 1.3.0.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/williamritchie/IRFinder/issues/97#issuecomment-647544457, or unsubscribe https://github.com/notifications/unsubscribe-auth/ADRQSTN5HY7CEGVOXKICQBDRX5RATANCNFSM4OBO27RQ .

dg520 commented 4 years ago

Hi @gabee-chan ,

OK. First check the current GCC you're using by gcc -v. Is it later than 4.9.0? If not, I think your cluster should have some mechanisms (e.g. module load) that allow you to load a later version of GCC. Load any GCC >=4.9.0 and that will solve half of your problem.

Then you need to manually compile IRFinder core, which you can find in the User Manual again here. Try to follow the instructions carefully. There are troubleshoots as well. If you encountered Error 2 and you cannot find libm.a or libm.so, you have to ask your system admin for the location of those files. If you encountered an error that is not described in the User Manual, attach here the full error message from the beginning to the end. Thank you.

Best, Dadi

gabee-chan commented 4 years ago

Hi Dadi!

Thank you very much for your help! I was able to run IRFinder in all my primates datasets. Still I have a last question. After doing the quantification step I'm getting as output both IRFinder-IR-dir.txt and IRFinder-IR-nondir.txt, I compared them and although they shared most of the coordinates the values of for example IRratio are different. I was wondering as the "non-dir" is the main output if I should use that.

Thank you very much for your help. Gaby

El mar., 23 jun. 2020 a las 14:44, Dadi (notifications@github.com) escribió:

Hi @gabee-chan https://github.com/gabee-chan ,

OK. First check the current GCC you're using by gcc -v. Is it later than 4.9.0? If not, I think your cluster should have some mechanisms (e.g. module load) that allow you to load a later version of GCC. Load any GCC >=4.9.0 and that will solve half of your problem.

Then you need to manually compile IRFinder core, which you can find in the User Manual again here https://github.com/williamritchie/IRFinder/wiki/Download-and-Install#compilation-optional. Try to follow the instructions carefully. There are troubleshoots as well. If you encountered Error 2 and you cannot find libm.a or libm.so, you have to ask your system admin for the location of those files. If you encountered an error that is not described in the User Manual, attach here the full error message from the beginning to the end. Thank you.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHub https://github.com/williamritchie/IRFinder/issues/97#issuecomment-647904060, or unsubscribe https://github.com/notifications/unsubscribe-auth/ADRQSTPZTRCIFOFC5MET2PLRYAXKTANCNFSM4OBO27RQ .

dg520 commented 4 years ago

Hi @gabee-chan ,

The difference between the two files are explained in the User Manual. Do you know whether your RNASeq is prepared with the awareness of strandness? You should ask wet-lab if you don't have that information.

Ideally, you should always stick with IRFinder-IR-dir.txt if it exists in your result folder, UNLESS you are 100% sure your RNASeq is not directional. IRFinder calculates the directionality for you internally. IRFinder-IR-dir.txt will never be genreated if IRFinder does not think your RNASeq is directional. But it's important you know this information from wet-lab beforehand, so that you can compare the truth with IRFinder's suggestion.

P.S. I strongly recommend you to read the User Manual. It contains details about how IRFinder works and has practical suggestions for users. If you don't understand any part, feel free to ask again.

Best, Dadi