Cuffquant errors after using HISAT2

Question: Cuffquant errors after using HISAT2

2.4 years ago by

ali.saberi92 • 0

ali.saberi92 • 0 wrote:

Hi,

I have 2 sequence reads archive and I want to do some RNA-Seq analysis on them.

Archive 1 : SRR1177960 (FastQ Files : SRR1177960_1, SRR1177960_2)

Archive 2 : SRR1177961 (FastQ Files : SRR1177961_1, SRR1177961_2)

I aligned both archives using HISAT2, and I got the results (BAM files).

Then I pass the results of each archive separately to Cufflinks, and I got the results (GTF files).

Then I pass the results to Cuffmerge, and I got the result (GTF file).

Then I pass the Cuffmerge result + HISAT2 result of archive 1 to the Cuffquant, and I got the result (CXB file).

BUT when I pass the Cuffmerge result + HISAT2 result of archive 2 to the Cuffquant, I got error

BAM record error: found spliced alignment without XS attribute

I attached the full text of error : cuffquant_error_archive2.txt

Can you help me?

(I forgot to say that I'm using Galaxy usegalaxy.org))

Thanks.

rna-seq galaxy cufflinks hisat2 cuffquant • 1.1k views

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modified 2.4 years ago by rkp21 • 20 • written 2.4 years ago by ali.saberi92 • 0

2.4 years ago by

rkp21 • 20

rkp21 • 20 wrote:

Hi,

I had a similar error message as yours after using HISAT2, but I had skipped the cuffquant step and gone directly to cuffdiff. See here: Cuffdiff Fatal error: Matched on error

The solution to the XS tag problem is that you need to find an option in HISAT2 that says "report alignments specifically tailored for cufflinks". I believe it was in advanced options. I think most likely both of your archives have this error since this error does not stop the cuffmerge or cuffdiff from running. If you click on info and then stderr you can view any errors that occurred while running the program.

I also had the same problem with loading the reference annotation, but I haven't found a solution to that yet. I will try using the iGenome transcriptome, recommended by Jen in the thread I linked to to, later today. Let me know if you find a good solution to this. Just out of curiosity, which reference transcriptome are you using? I am using hg19.

ADD COMMENT • link written 2.4 years ago by rkp21 • 20

Sorry for late reply, My problem solved few days ago, As you said, I use the --dta-cufflinks option (Report alignments tailored specifically for Cufflinks), and the problem solved! Thanks for your reply! I was working with Rnor6 (Rattus Norvegicus).

ADD REPLY • link written 2.4 years ago by ali.saberi92 • 0

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