skewer step is finished sh: 1: Syntax error: Bad fd number

[rashidma]

I am running docker4seq through 4SeqGUI in Ubuntu 20 LTS. I installed all the requirements in R as well as on command prompt.
I am using RNA seq pipeline. The indexing step is finished but i think this was not successful as I see some error Rout file.

Docker exit status: 0 

RSEM STAR index generation is finished
sh: 1: Syntax error: Bad fd number
b06b405b94f99b4c7541a0795121bdcce3647dfec37febffef2fc3c3450ac526
> 
> proc.time()
    user   system  elapsed 
   2.945    3.144 1819.779 

Please help to troubleshoot this. Also, how can I make sure that the indexing was successful??

Moreover when i run further step i got similar error at skewer step.
Routput_23_01_2024_17_04_48.Rout.txt

I attached R out file from the indexing step.

Thanks

[kendomaniac]

HI Mamood, sh: 1: Syntax error: Bad fd number this is an error tha refers to the use of of sh. You no not have to worry about it. Please try to run a mapping if you get the gtf_annotated_genes.results file everything is working smoothly. Please let me know if you get any problem. Best Raf From: Mamoon Rashid ***@***.***> Date: Monday, 29 January 2024 at 10:07 To: kendomaniac/docker4seq ***@***.***> Cc: Subscribed ***@***.***> Subject: [kendomaniac/docker4seq] skewer step is finished sh: 1: Syntax error: Bad fd number (Issue #6) I am running docker4seq through 4SeqGUI in Ubuntu 20 LTS. I installed all the requirements in R as well as on command prompt. I am using RNA seq pipeline. The indexing step is finished but i think this was not successful as I see some error Rout file. Docker exit status: 0 RSEM STAR index generation is finished sh: 1: Syntax error: Bad fd number b06b405b94f99b4c7541a0795121bdcce3647dfec37febffef2fc3c3450ac526

proc.time()

user system elapsed 2.945 3.144 1819.779 Please help to troubleshoot this. Also, how can I make sure that the indexing was successful?? Thanks — Reply to this email directly, view it on GitHub<#6>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/ACTBYA6H4LPLBB77UKXGXFDYQ5RDLAVCNFSM6AAAAABCPBSWFWVHI2DSMVQWIX3LMV43ASLTON2WKOZSGEYDIOJZGY3DINA>. You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[rashidma]

Hello Raffaele,
Thank you for your prompt reply.
I see genome.gtf file the genome folder with size 1.4 GB. I believe the indexing is ok.

Now, I started the "Genes, Isoforms counting RSEM". But it produces only "fastqc" output and the process finished.
Please see the R output file below:
Routput_29_01_2024_13_44_34.txt

Please help.
Thanks

[kendomaniac]

the index is too small. which is the organism you are interested? If Human or mouse you need 64Gb ram for the generation of the reference. If you tell me the organism I can generate it for you and provide a link for downloading it From: Mamoon Rashid ***@***.***> Date: Monday, 29 January 2024 at 11:55 To: kendomaniac/docker4seq ***@***.***> Cc: Raffaele Calogero ***@***.***>, Comment ***@***.***> Subject: Re: [kendomaniac/docker4seq] skewer step is finished sh: 1: Syntax error: Bad fd number (Issue #6) Hello Raffaele, Thank you for your prompt reply. I see genome.gtf file the genome folder with size 1.4 GB. I believe the indexing is ok. Now, I started the "Genes, Isoforms counting RSEM". But it produces only "fastqc" output and the process finished. Please see the R output file below: Routput_29_01_2024_13_44_34.txt<https://github.com/kendomaniac/docker4seq/files/14083331/Routput_29_01_2024_13_44_34.txt> Please help. Thanks — Reply to this email directly, view it on GitHub<#6 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/ACTBYA7MUNBMM2DBYVFO3JTYQ552PAVCNFSM6AAAAABCPBSWFWVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTSMJUGQ2DINRXGY>. You are receiving this because you commented.Message ID: ***@***.***>

[rashidma]

Please explain the problem in detail. You said the indexing is ok if I have the genome.gtf file.

I faced the problem in "skewer" as the Rout suggests.

Yes, I am using human genome. I really appreciate your help if you wanna provide the index for reference human genome.
I used the below links for genome and annotations:
ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz
ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz

Thank you once again.

[kendomaniac]

The problem is the generation of the reference, generally is better to use as assembly: https://ftp.ensembl.org/pub/release-111/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz and you need the gtf: https://ftp.ensembl.org/pub/release-111/gtf/homo_sapiens/Homo_sapiens.GRCh38.111.gtf.gz Homo_sapiens.GRCh38.dna.toplevel.fa.gz is very large and only contains extra information which are nearly of no interest if you work on transcriptomics. Top level includes all the particular insertions and reorganisation depicted by sequencing of multiple genomes. Those parts do not contain coding genes. The primary Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz requires more than 32 gb ram to be generated and top level much more. The problem is that STAR goes in out of memory and crashes when it runs out of memory. Unfortunately, you realize it only if you look inside the ended docker. The other way you realize it is that we are expecting nearly 34 Gb as size of the genome folder with the star reference sequence. The reason you get up to the trimmed fastq is because up to there it is not necessary the reference genome. Unfortunately, when star does not detect the correct structure of the genome it stops. I hope I was sufficiently clear, it is only a matter of RAM Cheers Raf From: Mamoon Rashid ***@***.***> Date: Tuesday, 30 January 2024 at 11:54 To: kendomaniac/docker4seq ***@***.***> Cc: Raffaele Calogero ***@***.***>, Comment ***@***.***> Subject: Re: [kendomaniac/docker4seq] skewer step is finished sh: 1: Syntax error: Bad fd number (Issue #6) Please explain the problem in detail. You said the indexing is ok if I have the genome.gtf file. I faced the problem in "skewer" as the Rout suggests. Yes, I am using human genome. I really appreciate your help if you wanna provide the index for reference human genome. I used the below links for genome and annotations: ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz Thank you once again. — Reply to this email directly, view it on GitHub<#6 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/ACTBYAYUOM7PCTAISODNOFTYRDGONAVCNFSM6AAAAABCPBSWFWVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTSMJWGU4DIMBVHE>. You are receiving this because you commented.Message ID: ***@***.***>

[rashidma]

Dear Raffaele,
Thank you for this great explanation.
As you suggested please generate a human reference from primary assembly for me. I will be very thankful to you.
Then i will run the mapping and counting container and test the pipeline.
Regards

[rashidma]

Hello Raffaele,
I managed to generate the reference using primary assembly. Also, I could run the mapping and counting. It looks like it is going fine.
Please see below files:

Routput_01_02_2024_10_08_28.txt

Thank you for your help
Mamoon

[kendomaniac]

It seems that everythings worked. If you have the gtf_annotated_genes.results file mapping was successful Cheers Raf From: Mamoon Rashid ***@***.***> Date: Thursday, 1 February 2024 at 11:10 To: kendomaniac/docker4seq ***@***.***> Cc: Raffaele Calogero ***@***.***>, Comment ***@***.***> Subject: Re: [kendomaniac/docker4seq] skewer step is finished sh: 1: Syntax error: Bad fd number (Issue #6) Hello Raffaele, I managed to generate the reference using primary assembly. Also, I could run the mapping and counting. It looks like it is going fine. Please see below files: Routput_01_02_2024_10_08_28.txt<https://github.com/kendomaniac/docker4seq/files/14123481/Routput_01_02_2024_10_08_28.txt> Thank you for your help Mamoon — Reply to this email directly, view it on GitHub<#6 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/ACTBYA4EUF5ZSKMOW6TIP2LYRNSZZAVCNFSM6AAAAABCPBSWFWVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMYTSMRQHE3DMOJRGQ>. You are receiving this because you commented.Message ID: ***@***.***>