[kaus]

Same issue

Hi guys,

this is probably a good place to ask this question. But I ran STAR with that option to add the XS attribute. But when I run cufflinks I get this

BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
BAM record error: found spliced alignment without XS attribute
> Processing Locus chr1:2835641-2836005 [ ] 0%BAM record error: found spliced alignment without XS attribute
> Processing Locus chr1:2882539-2882944 [ ] 0%BAM record error: found spliced alignment without XS attribute
> Processing Locus chr1:3536416-3537049 [ ] 0%BAM record error: found spliced alignment without XS attribute
> Processing Locus chr1:3537448-3539171 [ ] 0%BAM record error: found spliced alignment without XS attribute
> Processing Locus chr1:3646678-3647038 [ ] 0%BAM record error: found spliced alignment without XS attribute

It does keep running, and the percentage does increase, but I was wondering if this behaviour is normal for STAR and cufflinks?

Hey guys
I'm having the same issue. When I run Cufflinks this is what I get
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File /Users/sohailkhan/Documents/MiSeq_data/12082014_CLIP/untag_sorted doesn't appear to be a valid BAM file, trying SAM...
[12:24:10] Loading reference annotation.
[12:24:11] Inspecting reads and determining fragment length distribution.
> Processing Locus AB325691:1478-3197 [ ] 0%SAM error on line 49: found spliced alignment without XS attribute
> Processing Locus I:27352-27763 [**** ] 16%SAM error on line 380: found spliced alignment without XS attribute
SAM error on line 381: found spliced alignment without XS attribute
SAM error on line 384: found spliced alignment without XS attribute
> Processing Locus I:29763-31069 [**** ] 16%SAM error on line 474: found spliced alignment without XS attribute
SAM error on line 479: found spliced alignment without XS attribute
SAM error on line 480: found spliced alignment without XS attribute
SAM error on line 481: found spliced alignment without XS attribute
SAM error on line 482: found spliced alignment without XS attribute
SAM error on line 485: found spliced alignment without XS attribute
SAM error on line 486: found spliced alignment without XS attribute
SAM error on line 488: found spliced alignment without XS attribute
SAM error on line 489: found spliced alignment without XS attribute
> Processing Locus I:45874-48399 [**** ] 16%SAM error on line 603: found spliced alignment without XS attribute
SAM error on line 604: found spliced alignment without XS attribute
I used the latest STAR release for Mac and I'm using the latest release of Cufflinks. The STAR options I used were:

--runThreadN 6 --outSJfilterOverhangMin 30 10 10 10
--outFilterMismatchNmax 6 --alignIntronMin 10 --alignIntronMax 500 --alignMatesGapMax 500 --alignSplicedMateMapLmin 1 --outFilterType BySJout --outFilterIntronMotifs RemoveNoncanonical --sjdbGTFfile /PATH --sjdbOverhang 74
Is there any option I need to add for the right XS attributes?

[alexdobin]

Hey guys
I'm having the same issue. When I run Cufflinks this is what I get
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File /Users/sohailkhan/Documents/MiSeq_data/12082014_CLIP/untag_sorted doesn't appear to be a valid BAM file, trying SAM...
[12:24:10] Loading reference annotation.
[12:24:11] Inspecting reads and determining fragment length distribution.
> Processing Locus AB325691:1478-3197 [ ] 0%SAM error on line 49: found spliced alignment without XS attribute
> Processing Locus I:27352-27763 [**** ] 16%SAM error on line 380: found spliced alignment without XS attribute
SAM error on line 381: found spliced alignment without XS attribute
SAM error on line 384: found spliced alignment without XS attribute
> Processing Locus I:29763-31069 [**** ] 16%SAM error on line 474: found spliced alignment without XS attribute
SAM error on line 479: found spliced alignment without XS attribute
SAM error on line 480: found spliced alignment without XS attribute
SAM error on line 481: found spliced alignment without XS attribute
SAM error on line 482: found spliced alignment without XS attribute
SAM error on line 485: found spliced alignment without XS attribute
SAM error on line 486: found spliced alignment without XS attribute
SAM error on line 488: found spliced alignment without XS attribute
SAM error on line 489: found spliced alignment without XS attribute
> Processing Locus I:45874-48399 [**** ] 16%SAM error on line 603: found spliced alignment without XS attribute
SAM error on line 604: found spliced alignment without XS attribute
I used the latest STAR release for Mac and I'm using the latest release of Cufflinks. The STAR options I used were:

--runThreadN 6 --outSJfilterOverhangMin 30 10 10 10
--outFilterMismatchNmax 6 --alignIntronMin 10 --alignIntronMax 500 --alignMatesGapMax 500 --alignSplicedMateMapLmin 1 --outFilterType BySJout --outFilterIntronMotifs RemoveNoncanonical --sjdbGTFfile /PATH --sjdbOverhang 74
Is there any option I need to add for the right XS attributes?

Hi @kaus,

to add XS flag, you need the --outSAMstrandField intronMotif option which was mentioned before. Note that --sjdbGTFfile /PATH --sjdbOverhang 74 options have to be used at the genome generation step, at the mapping stage they have no effect.

Cheers
Alex

[tonup69]

Cuffdiff fails using STAR mapped BAM files.

Hi folks,

I think this would be a good place to post my current issue since it must have something to do with using STAR and Cufflinks. I have now had two fatal errors in my analysis (I may have solved one of them, but it may have caused the second one):

1) Cuffmerge failed because it indicated that there were duplicate IDs in the gtf file. We figured out that this was because transcripts and exons can have the same cords (especially in the fly genome). We fixed this by eliminating all transcript lines and leaving only exon level information in the file. A similar fix was posted here, but I can't find the link right now.

2) Cuffdiff now throws an error for the BAM files:

Fatal error: Error
[05:15:57] Loading reference annotation.
[05:16:02] Inspecting maps and determining fragment length distributions.
BAM record error: found spliced alignment without XS attribute

I set up STAR to run using the "add XS for Cufflinks" option, but I guess that didn't work since I got this Cuffdiff error AND a quick look at the BAM files did not reveal any lines with the XS tag.

I am running this analysis on a local install of GALAXY. I do not code or use the command line, but I have a bioinformaticist who can help me if I need to manipulate the files outside of GALAXY.

P.S. I know I could use Tophat for mapping, but STAR gives better results with Ion Proton data (we tested in house). Also, do you think this could be due to using the "all modern SAM tools" attribute and not the "original SAM tools"

Sorry for the long message - I need to analyze this data by Monday so I can put a figure in my grant due next week!

LTR

[alexdobin]

Hi LTR,

the "--outSAMstrandField intronMotif" option should produce XS flags for all spliced alignments. If you used this option and Cufflinks still complains about it, please send me the Log.out and SAM/BAM file.

Cheers
Alex

[natallah]

ran star on stranded data, still getting Cufflinks XS attributes error

Hi I ran STAR on stranded data with this command:
STAR --runThreadN 4 --genomeDir ./STARoutput --readFilesIn OS1.common_1.fastq OS1.common_2.fastq --outFileNamePrefix ./OS1trimmed

However when I try running Cufflinks after I get the following error:
BAM record error: found spliced alignment without XS attribute

The version is:

versionSTAR 020201


Thanks,
Nadia

[alexdobin]

Hi I ran STAR on stranded data with this command:
STAR --runThreadN 4 --genomeDir ./STARoutput --readFilesIn OS1.common_1.fastq OS1.common_2.fastq --outFileNamePrefix ./OS1trimmed

However when I try running Cufflinks after I get the following error:
BAM record error: found spliced alignment without XS attribute

The version is:

versionSTAR 020201


Thanks,
Nadia

Hi Nadia,

if you your RNA-seq data is stranded, you need to run Cufflinks with --library-type option. If your RNA-seq data is unstranded, you have to run STAR with "--outSAMstrandField intronMotif" option.

Cheers
Alex