Question: HISAT2-StringTie-Cuffdiff error: BAM record error: found spliced alignment without XS attribute
0
gravatar for jolin0701-dy
2.3 years ago by
jolin0701-dy70
jolin0701-dy70 wrote:

I know Hisat2-StringTie-ballgown pipeline is very useful, but I'd also like to set up a Hisat2-StringTie-Cuffdiff pipeline.

Here are the my commands for each step:

$hisat2 --dta -x fruitfly_tran -1 smGSM794483_C1_R1_1.fq -2 smGSM794483_C1_R1_2.fq -S smGSM794483_C1_R1.sam

$samtools view -bS smGSM794483_C1_R1.sam > smGSM794483_C1_R1_unsorted.bam

$samtools sort smGSM794483_C1_R1_unsorted.bam smGSM794483_C1_R1

$stringtie -G fruitfly.gtf -o smGSM794483_C1_R1.gtf -l smGSM794483_C1_R1 smGSM794483_C1_R1.bam

$gffcompare -r fruitfly.gtf -G -o merged stringtie_merged.gtf

$cuffdiff -o cuffdiff -b fruitfly.fa -p 4 -L C1,C2 -u stringtie_merged.gtf smGSM794483_C1_R1.bam,smGSM794484_C1_R2.bam,smGSM794485_C1_R3.bam smGSM794486_C2_R1.bam,smGSM794487_C2_R2.bam,smGSM794488_C2_R3.bam

And in the final step of cuffdiff,

I saw some errors:

BAM record error: found spliced alignment without XS attribute

I noticed that some parameters in the pipeline may be not good for cuffdiff.

In the hisat2, I should use --dta-cufflinks other than --dta ...

Would you please give me some suggestions?

Thanks so much for your help!!

rna-seq • 3.0k views
ADD COMMENTlink modified 2.3 years ago by Macspider2.8k • written 2.3 years ago by jolin0701-dy70
2
gravatar for Macspider
2.3 years ago by
Macspider2.8k
Vienna - BOKU
Macspider2.8k wrote:

$hisat2 --dta -x fruitfly_tran -1 smGSM794483_C1_R1_1.fq -2 smGSM794483_C1_R1_2.fq -S smGSM794483_C1_R1.sam

Modify --dta in --dta-cufflinks, and use Cuffdiff directly without Stringtie for an immediate result.

Otherwise, run hisat2 again with --dta-cufflinks and then use in Cuffdiff:

  • the GTF file obtained from the hisat2 run with just --dta
  • the BAM files obtained from the hisat2 run with --dta-cufflinks

For an overall overview of this workflow, you can see it like this:

  1. Run 2 parallel HISAT2 runs on the same files: one with --dta and one with --dta-cufflinks
  2. Run Stringtie on the BAM files obtained from the --dta mode
  3. Run Cuffdiff with the GTF file obtained from Stringtie, and the BAM files obtained from the --dta-cufflinks mode

This way you should be sure that Cuffdiff does the job silently.

ADD COMMENTlink written 2.3 years ago by Macspider2.8k

Thanks so much for your detailed information......

ADD REPLYlink written 2.3 years ago by jolin0701-dy70

What I didn't write is that the issue about the XS tag is famous with Cufflinks and programs from the Johns Hopkins University. Some links:

ADD REPLYlink written 2.3 years ago by Macspider2.8k

I met the same error and learned so much from your answers. But a question still remains to me: I run HISAT2 with the option -dta-cufflinks, then I run STRINGTIE on the BAM files obtained from the -dta-cufflinks mode, and the following cuffcompare and cuffdiff, no error occured. So Why should run 2 parallel HISAT2 that one with -dta and one with -dta-cufflinks? Is there any difference between the GTF files generated from STRINGTIE if one with -dta and one with -dta-cufflinks? Thanks a lot!

ADD REPLYlink written 2.1 years ago by lxchen0
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