tophat/cufflinks error

[peromhc]

Hi All,

I'm having a reoccurance of the "Error: this SAM file doesn't appear to be correctly sorted!" issue..

tophat 1.2.0 and cufflinks 1.0.2

bam file created using tophat

/home/matthew/tophat-1.2.0/tophat -o /media/hd/annotation/tophat/social -r 100 -p 8 --allow-indels --library-type fr-unstranded /media/hd/annotation/tophat/bowtie.index.26May11.tuco.mrna /media/hd/tuco/mrna/trimmed/apache.final.clip_1.fq,/media/hd/tuco/mrna/trimmed/ep.final.clip_1.fq /media/hd/tuco/mrna/trimmed/apache.final.clip_2.fq,/media/hd/tuco/mrna/trimmed/ep.final.clip_2.fq

In cufflinks:

/home/matthew/cufflinks-1.0.2/cufflinks -o /media/hd/annotation/tophat/solitary/cufflinks --num-importance-samples 5000 --max-mle-iterations 20000 -p 16 /media/hd/annotation/tophat/solitary/accepted_hits.bam
You are using Cufflinks v1.0.2, which is the most recent release.
[18:04:56] Inspecting reads and determining fragment length distribution.
> Processing Locus 10034_gs536_ge1663_3u1664_E [                         ]   0%
Error: this SAM file doesn't appear to be correctly sorted!
	current hit is at 10036_gs2_ge544_3u545_GENSCAN00000013565_Pteropus_neurexin:0, last one was at 10035_gs2_ge1360_ENSPTRP00000059044_PREDICTED::1331
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.

[fgh1124]

revise the script!

http://seqanswers.com/forums/showthread.php?p=42800#post42800

[peromhc]

yeah, I think this something different.. I have a BAM file created in tophat.. The referenced post had to do with a GTF file created in cufflinks.

[fgh1124]

I am sorry that I misunderstood you qeustion. I guess your problem maybe is the samtools. You can check if it work correctly!

[peromhc]

not sure, I am using samtools 0.1.16.

The samtools sort command looks right..

[peromhc]

Any ideas about this?? I get the same error with the new tophat 1.3.0..

[peromhc]

here are a few lines from the .sam


head -n 30 /media/hd/annotation/tophat/solitary/accepted_hits.sam

@HD VN:1.0 SO:coordinate
@RG ID:tuco SM:solitary PI:100
@SQ SN:10000_mitochondrial.carrie LN:1756
@SQ SN:10002_DET1-.and.DDB1-assoc LN:1891
@SQ SN:10003_DET1.and.DDB1.associ LN:1282
@SQ SN:10005_polymerase.(DNA-dire LN:1432
@SQ SN:10006_DET1-.and.DDB1-assoc LN:1943
@SQ SN:10007_microRNA.1909. LN:856
@SQ SN:10009_chromatin.modifying. LN:760
@SQ SN:1000_Ubiquinol-cytochrome LN:624
@SQ SN:10010_Notch.homolog.3.(Dro LN:459
@SQ SN:10011_diacylglycerol.kinas LN:2191
@SQ SN:10013_Ubiquitin-related.mo LN:852
@SQ SN:10016_chibby.homolog.1.(Dr LN:793
@SQ SN:10017_metastasis.associate LN:518
@SQ SN:10019_Nucleoside.diphospha LN:477
@SQ SN:1001_histone.deacetylase. LN:3124
@SQ SN:10020_non-metastatic.cells LN:969
@SQ SN:10021_Nucleoside.diphospha LN:494
@SQ SN:10023_Nucleoside.diphospha LN:698
@SQ SN:10024_SLC2A4.regulator. LN:457
@SQ SN:10025_chondroitin.polymeri LN:1440
@SQ SN:10029_annexin.A6. LN:1845
@SQ SN:1002_DEAH.(Asp-Glu-Ala-As LN:1343
@SQ SN:10030_integrin-linked.kina LN:1263
@SQ SN:10032_Ca++-dependent.secre LN:2067
@SQ SN:10033_integrin-linked.kina LN:1159
@SQ SN:10034_Neurexin-1-alpha.Pre LN:1876
@SQ SN:10035_PREDICTED:.Pan.trogl LN:1362

[emilyjia2000]

I had similar problem before, I used Samtools sort the BAM files

[woodydon]

Try samtools 0.1.8.