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Old 03-21-2010, 11:18 PM   #1
seq_GA
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Default Cufflinks with solexa data

Has anyone used cufflinks for solexa RNA-Seq data?

Te reads are mapped using eland_rna algorithm and used export2sam.pl to convert the eland mapping into sam format.

I get the following error when I try to use cufflinks.

Code:
$ ./cufflinks /test/lib.sam 
Counting hits in map
Error: this SAM file doesn't appear to be correctly sorted!
        current hit is at RM:-1, last one was at (null):0
Please let me know the steps involved from solexa eland mapping data. Thanks.
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Old 03-21-2010, 11:53 PM   #2
kopi-o
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I would assume that the SAM file is indeed unsorted, as the error message says. If so, you can use the samtools package to sort the SAM file (I think you'll first have to convert it to BAM using "samtools view" before sorting, and then convert back) and try again.

Alternatively (and easier), you can sort the Eland output file (or the SAM file) on the command line using a command like

sort -o infile.bed -k 1,1 -k 2,2n infile.bed

(if the chromosome is in column 1 and the starting coordinate is in column 2, that is ... if not, you have to substitute the correct column numbers for "1" and "2" above)
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Old 03-22-2010, 08:36 AM   #3
middlemale
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possibly your sam file requires sorted by samtools . I did not have experience. good luck.
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Old 03-22-2010, 01:57 PM   #4
DrD2009
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Use the following command to sort your SAM files. I experienced the same problem and this fixed it. The code is from the Cufflinks manual, no need for samtools or anything else.

Code:
sort -k 3,3 -k 4,4n hits.sam > hits.sam.sorted
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Old 03-23-2010, 01:39 AM   #5
seq_GA
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Hi
Thanks for the response. I did sort the sam file as per the manual but I still get the error as below:

Code:
Counting hits in map
Error: this SAM file doesn't appear to be correctly sorted!
        current hit is at mm9:-43, last one was at (null):0
My reference genome chromosome names are c1,c2,c3...etc and splice junction reads starts with mm9. This is according to the eland mappingwhich includes mapping data against genome which includes c1,c2,c3 etc.. and the splice juctions generated (mm9)

Please let me know how to overcome this problem. Thanks.
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Old 03-23-2010, 05:16 AM   #6
Thomas Doktor
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I would try aligning the reads with TopHat instead. I have no experience with the Eland RNA-seq alignment program, but I doubt TopHat would do a much worse job.
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Old 05-13-2010, 02:57 AM   #7
seq_GA
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Hi,
HAs anyone tried converting solexa eland output into sam format and use cufflinks successfully? I would erally appreciate very much for any help. Thanks.
Regards
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