Error when launching star : EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem

[Marianne Burbage]

Hello everyone,

I am encoutering the following error when launching STAR:

"EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem"

The star Log/out file is attached.

Do you have an idea what could be the issue?

Thank you very much for your help!

Marianne

[Alexander Dobin]

Hi Marianne,

the Log.out file did not get attached, I am afraid.

Thanks!

Alex

[Marianne Burbage]

Hello Alex,

I have repeated errors when trying to send the attachment.

Here's a link for the log file.

https://www.dropbox.com/s/srxy5uktzq63rcg/test_SRX864933_1Log.out?dl=0

[Marianne Burbage]

Hello Alex,

I tried some optimisation but still run into the same issue. Here is the tail of my log.out file:

"Shared memory is not used for genomes. Allocated a private copy of the genome.
Genome file size: 2798470136 bytes; state: good=1 eof=0 fail=0 bad=0
Loading Genome ... done! state: good=1 eof=0 fail=0 bad=0; loaded 2798470136 bytes
SA file size: 22355261861 bytes; state: good=1 eof=0 fail=0 bad=0
Loading SA ... done! state: good=1 eof=0 fail=0 bad=0; loaded 22355261861 bytes
Loading SAindex ... done: 1565873619 bytes
Finished loading the genome: Tue Apr 27 19:05:07 2021

Apr 27 19:05:07 ..... inserting extra sequences into genome indexes

EXITING because of FATAL ERROR: cannot insert sequence on the fly because of strand GstrandBit problem

SOLUTION: please contact STAR author at https://groups.google.com/forum/#!forum/rna-star

Apr 27 19:05:42 ...... FATAL ERROR, exiting"

Thanks so much for your help!
Marianne

[Alexander Dobin]

Hi Marianne,

please remove --genomeFastaFiles /shared/bank/mus_musculus/mm10/fasta/mm10.fa from the command line at the mapping stage.

Since you specified these sequences at the genome generation step, you do not need to specify them again at the mapping stage.

Another point, not related to this issue - I highly recommend using a GTF file with transcript annotations at the genome generation, as it improves the accuracy of the alignments.

Cheers

Alex

[Marianne Burbage]

Hello Alex,

Thank you very much for your answer, it has indeed worked after that.

Regarding the gtf, I had the impression that the one I used had transcript information, but I might be mistaken? Attached are the first lines of the gtf I used.

1    havana    gene    3073253    3074322    .    +    .    gene_id "ENSMUSG00000102693"; gene_version "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC";
1    havana    transcript    3073253    3074322    .    +    .    gene_id "ENSMUSG00000102693"; gene_version "1"; transcript_id "ENSMUST00000193812"; transcript_version "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC"; transcript_name "4933401J01Rik-201"; transcript_source "havana"; transcript_biotype "TEC"; tag "basic"; transcript_support_level "NA";
1    havana    exon    3073253    3074322    .    +    .    gene_id "ENSMUSG00000102693"; gene_version "1"; transcript_id "ENSMUST00000193812"; transcript_version "1"; exon_number "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC"; transcript_name "4933401J01Rik-201"; transcript_source "havana"; transcript_biotype "TEC"; exon_id "ENSMUSE00001343744"; exon_version "1"; tag "basic"; transcript_support_level "NA";
1    ensembl    gene    3102016    3102125    .    +    .    gene_id "ENSMUSG00000064842"; gene_version "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA";
1    ensembl    transcript    3102016    3102125    .    +    .    gene_id "ENSMUSG00000064842"; gene_version "1"; transcript_id "ENSMUST00000082908"; transcript_version "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA"; transcript_name "Gm26206-201"; transcript_source "ensembl"; transcript_biotype "snRNA"; tag "basic"; transcript_support_level "NA";
1    ensembl    exon    3102016    3102125    .    +    .    gene_id "ENSMUSG00000064842"; gene_version "1"; transcript_id "ENSMUST00000082908"; transcript_version "1"; exon_number "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA"; transcript_name "Gm26206-201"; transcript_source "ensembl"; transcript_biotype "snRNA"; exon_id "ENSMUSE00000522066"; exon_version "1"; tag "basic"; transcript_support_level "NA";
1    ensembl_havana    gene    3205901    3671498    .    -    .    gene_id "ENSMUSG00000051951"; gene_version "5"; gene_name "Xkr4"; gene_source "ensembl_havana"; gene_biotype "protein_coding";
1    havana    transcript    3205901    3216344    .    -    .    gene_id "ENSMUSG00000051951"; gene_version "5"; transcript_id "ENSMUST00000162897"; transcript_version "1"; gene_name "Xkr4"; gene_source "ensembl_havana"; gene_biotype "protein_coding"; transcript_name "Xkr4-203"; transcript_source "havana"; transcript_biotype "lncRNA"; transcript_support_level "1";
1    havana    exon    3213609    3216344    .    -    .    gene_id "ENSMUSG00000051951"; gene_version "5"; transcript_id "ENSMUST00000162897"; transcript_version "1"; exon_number "1"; gene_name "Xkr4"; gene_source "ensembl_havana"; gene_biotype "protein_coding"; transcript_name "Xkr4-203"; transcript_source "havana"; transcript_biotype "lncRNA"; exon_id "ENSMUSE00000858910"; exon_version "1"; transcript_support_level "1";
1    havana    exon    3205901    3207317    .    -    .    gene_id "ENSMUSG00000051951"; gene_version "5"; transcript_id "ENSMUST00000162897"; transcript_version "1"; exon_number "2"; gene_name "Xkr4"; gene_source "ensembl_havana"; gene_biotype "protein_coding"; transcript_name "Xkr4-203"; transcript_source "havana"; transcript_biotype "lncRNA"; exon_id "ENSMUSE00000866652"; exon_version "1"; transcript_support_level "1";

Many thanks for your precious help!

Cheers,

Marianne

[Alexander Dobin]

Hi Marianne,

the GTF looks good, but you need to specify it with --sjdbGTFfile ann.gtf at the genome generation step.

Cheers

Alex