cufflinks running problem

[camelbbs]

Hi,

I am using bowtie and cufflinks to get gene expression from RNA seq data.

My workflow is like this:

bowtie -p 4 --best --strata -m 1 --sam mm9/mm9 -q SRR032476.fastq SRR032476.sam

and when i run

cufflinks SRR032476.sam

I got an error like

cufflinks: /usr/lib64/libz.so.1: no version information available (required by cufflinks)
[bam_header_read] EOF marker is absent.
File SRR032476.sam doesn't appear to be a valid BAM file, trying SAM...
[15:46:53] Inspecting reads and determining fragment length distribution.
> Processing Locus chr2:131113838-131113873    [                         ]   0%
Error: this SAM file doesn't appear to be correctly sorted!
	current hit is at chr1:162968481, last one was at chr9:108470655
Cufflinks requires that if your file has SQ records in
the SAM header that they appear in the same order as the chromosomes names 
in the alignments.
If there are no SQ records in the header, or if the header is missing,
the alignments must be sorted lexicographically by chromsome
name and by position.

the head of sam file is:

@HD	VN:1.0	SO:unsorted
@SQ	SN:chr1	LN:197195432
@SQ	SN:chr2	LN:181748087
@SQ	SN:chr3	LN:159599783
@SQ	SN:chr4	LN:155630120
@SQ	SN:chr5	LN:152537259
@SQ	SN:chr6	LN:149517037
@SQ	SN:chr7	LN:152524553
@SQ	SN:chr8	LN:131738871
@SQ	SN:chr9	LN:124076172
@SQ	SN:chr10	LN:129993255
@SQ	SN:chr11	LN:121843856
@SQ	SN:chr12	LN:121257530
@SQ	SN:chr13	LN:120284312
@SQ	SN:chr14	LN:125194864
@SQ	SN:chr15	LN:103494974
@SQ	SN:chr16	LN:98319150
@SQ	SN:chr17	LN:95272651
@SQ	SN:chr18	LN:90772031
@SQ	SN:chr19	LN:61342430
@SQ	SN:chrX	LN:166650296
@SQ	SN:chrY	LN:15902555
@SQ	SN:chrM	LN:16299
@PG	ID:Bowtie	VN:0.12.7	CL:"bowtie -p 4 --best --strata -m 1 --sam mm9/mm9 -q SRR032476.fastq SRR032476.sam"
SRR032476.4 I354_2_FC30605AAXX:2:1:2:1524 length=35	4	*	0	0	**	0	0	NGAGGTAGTAGGTTGTATAGTTATCGTATTCCGTT	!IIIIIIIIIIIIIIIIII9IIIII,III+IB0H$	XM:i:0

Anyone can help this? Thanks so much.

[blue mood]

Any suggestions?

[hlwright]

Hi
have you tried the sort function as described in the cufflinks manual?

"The SAM file supplied to Cufflinks must be sorted by reference position. If you aligned your reads with TopHat, your alignments will be properly sorted already. If you used another tool, you may want to make sure they are properly sorted as follows:

sort -k 3,3 -k 4,4n hits.sam > hits.sam.sorted "

[darked89]

Any suggestions?

Get the newest version of (samtools 0.1.17), and convert the SAM to sorted BAM, preferably with index.

[kwatts59]

http://i.seqanswers.com/questions/159/sorting-of-sam-file-generated-by-novoalign

[blue mood]

Get the newest version of (samtools 0.1.17), and convert the SAM to sorted BAM, preferably with index.

Thanks,it works well.

[blue mood]

i wonder if there is any way to avoid this issue?