cufflinks large bundles warning

Question

[Sam]

Dear All

I used star to align the PE lib with this command and then I converted sam file to bam and sorted it with samtools but after running the cufflinks I got "Warning: Skipping large bundle" how I can fix it ?

Thanks

STAR --runThreadN 12 --genomeDir  /genome_index_star -sjdbGTFfile /genome_index/Egene_exons.gtf --readFilesIn R1.fastq,R2.fastq --outFileNamePrefix test  --outSAMstrandField intronMotif

cufflinks -o cufflink_out_gtf -g ./Egene_exons.gtf test_Aligned.out_sorted.bam

[Bastien Hervé]

Try to increase the --max-bundle-length in cufflinks command, try 10000000

similar error

cufflinks options

[Sam]

but I tried also(the same lib ) with Tophat2 and Hisat2 and cufflinks worked well without any warning and error. should I change any flag in the star to be compatible with cufflinks?

[Bastien Hervé]

In STAR manual

In addition, it is recommended to remove the non-canonical junctions for Cufflinks runs using --outFilterIntronMotifs RemoveNoncanonical.

jM jI attributes require samtools 0.1.18 or later, and were reported to be incompatible with some downstream tools such as Cufflinks.

I would say also that what you got is just a warning not an error

[Sam]

Thanks for your help, but with this warning, the processing time of cufflinks increased at least 10 times.

[Bastien Hervé]

Did you try the --max-bundle-length and the --outFilterIntronMotifs RemoveNoncanonical ?