Question about running Cufflinks on ENCODE RNA-Seq data

juliacc · 08-15-2013, 03:39 AM

Hello,

I'm new in the community but I found many helpful posts in the past, so here is my question.

I downloaded around 60 .bam files from the ENCODE repository. The header size was giving problems and I converted them to .sam as one of the answers in these thread suggested.

Code:

samtools view -h -o file.sam file.bam

And when I run Cufflinks I get many lines with this error:

SAM error on line 862765: found spliced alignment without XS attribute

I am executing this on batch, so it is still running and, for some files, Cufflinks finish and produces the gene count, for other files I am not sure if it just hasn't finished yet.

I've been reading many posts about the XS attribute, but I don't know why would it be missing if I just converted from .bam to .sam. And would you know if it affects the read counts for genes?

Thank you,
Julia

AmitPande · 11-06-2013, 07:10 AM

Did you find a solution yet ? Even I am facing the same prolem with ENCODE RNAseq data

bye · 11-06-2013, 11:39 AM

Not sure how this error will affect the counts, but it's not hard to add XS tags.

Code:

samtools view -Xh yourfile.bam | awk -F"\t" '{if(NF<7)print $0; if($2~"P"&&$2!~"r"){print $0"\tXS:A:+"}else if($2~"P"&&$2~"r"){print $0"\tXS:A:-"}else{print $0}}' > yourfile.xs.sam

XS tags label the strand of alignment; samtools view -X will convert the flags into human readable format, then you can add strand information accordingly.

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08-15-2013, 03:39 AM	#1
juliacc Junior Member Location: Finland Join Date: Aug 2013 Posts: 1	Question about running Cufflinks on ENCODE RNA-Seq data Hello, I'm new in the community but I found many helpful posts in the past, so here is my question. I downloaded around 60 .bam files from the ENCODE repository. The header size was giving problems and I converted them to .sam as one of the answers in these thread suggested. Code: samtools view -h -o file.sam file.bam And when I run Cufflinks I get many lines with this error: SAM error on line 862765: found spliced alignment without XS attribute I am executing this on batch, so it is still running and, for some files, Cufflinks finish and produces the gene count, for other files I am not sure if it just hasn't finished yet. I've been reading many posts about the XS attribute, but I don't know why would it be missing if I just converted from .bam to .sam. And would you know if it affects the read counts for genes? Thank you, Julia

11-06-2013, 11:39 AM	#3
bye Junior Member Location: NJ Join Date: Sep 2010 Posts: 8	Not sure how this error will affect the counts, but it's not hard to add XS tags. Code: samtools view -Xh yourfile.bam \| awk -F"\t" '{if(NF<7)print $0; if($2~"P"&&$2!~"r"){print $0"\tXS:A:+"}else if($2~"P"&&$2~"r"){print $0"\tXS:A:-"}else{print $0}}' > yourfile.xs.sam XS tags label the strand of alignment; samtools view -X will convert the flags into human readable format, then you can add strand information accordingly.

11-06-2013, 07:10 AM	#2
AmitPande Junior Member Location: India Join Date: Dec 2011 Posts: 6	Did you find a solution yet ? Even I am facing the same prolem with ENCODE RNAseq data