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Old 01-19-2013, 07:20 AM   #1
syintel87
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Location: Universe

Join Date: Dec 2012
Posts: 81
Default ERROR: cufflinks, cuffmerge, cuffdiff

bowtie2-build Mh.fa Mh &

tophat2 -p 8 -o t1_Mh -G Mh.gff Mh t1.1_fq,t1.2_fq,t1.3_fq,t1.4_fq,t1.5_fq 2>> tophat.log &
tophat2 -p 8 -o t2_Mh -G Mh.gff Mh t2.1_fq,t2.2_fq,t2.3_fq,t2.4_fq,t2.5_fq 2>> tophat.log &

===============no problem until here=================

samtools view -o accepted_hits.sam accepted_hits.bam
cufflinks -o cufflinks_out t1_accepted_hits.sam 2>> cufflinks.log &

You are using Cufflinks v2.0.2, which is the most recent release.
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File IR_t1_Mh/accepted_hits.sam doesn't appear to be a valid BAM file, trying SAM...
[10:52:43] Inspecting reads and determining fragment length distribution.
> Processed 9677 loci. [*************************] 100%
> Map Properties:
> Normalized Map Mass: 11715284.00
> Raw Map Mass: 11715284.00
> Fragment Length Distribution: Truncated Gaussian (default)
> Default Mean: 200
> Default Std Dev: 80
[10:53:42] Assembling transcripts and estimating abundances.


output files:
-genes.fpkm_tracking,
-isoforms.fpkkm_tracking,
-skipped.gtf,
-and transcripts.gtf.

Cufflinks worked when sam file was used and gff file was dropped.

===============no problem until here=================

cufflinks -g Mh.gff -o cufflinks_out t1_accepted_hits.sam

You are using Cufflinks v2.0.2, which is the most recent release.
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File IR_t1_Mh/accepted_hits.sam doesn't appear to be a valid BAM file, trying SAM...
[10:59:30] Loading reference annotation.
[10:59:31] Inspecting reads and determining fragment length distribution.
> Processing Locus Mh:3451:MhA1_Contig3451:113 [************************ ] 99%
Error: this SAM file doesn't appear to be correctly sorted!
current hit is at Mh:0004:MhA1_Contig4:380, last one was at Mh:0003:MhA1_Contig3:890
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.


Cufflinks didn't work when gff file was included.
It seemed that cufflinks stoppted at Contig3.

===============problem occurred above=================

cufflinks -o cufflinks_out t1_accepted_hits.bam

You are using Cufflinks v2.0.2, which is the most recent release.
Error: sort order of reads in BAMs must be the same


Cufflinks didn't work when bam file was used and gff file was dropped.

===============problem occurred above=================

cufflinks -g Mt.gff -o cufflinks_out t1_accepted_hits.bam 2>> cufflinks.log &

You are using Cufflinks v2.0.2, which is the most recent release.
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File IR_t1_Mh/accepted_hits.sam doesn't appear to be a valid BAM file, trying SAM...
[10:50:49] Loading reference annotation.
[10:50:50] Inspecting reads and determining fragment length distribution.
> Processing Locus Mh:3451:MhA1_Contig3451:113 [************************ ] 99%
Error: this SAM file doesn't appear to be correctly sorted!
current hit is at Mh:0004:MhA1_Contig4:380, last one was at Mh:0003:MhA1_Contig3:890
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.


Cufflinks didn't work when bam file and gff file were used.
It seemed that cufflinks stoppted at Contig3.

===============problem occurred above=================

cuffmerge -o merge_IR_Mt -g Mt.gff IR_Mt_transcript.txt 2>> cuffmerge.log &

[Sat Jan 19 11:10:13 2013] Preparing output location merge_IR_Mh/
[Sat Jan 19 11:10:15 2013] Converting GTF files to SAM
[11:10:15] Loading reference annotation.
[11:10:17] Loading reference annotation.
[11:10:18] Loading reference annotation.
[11:10:18] Loading reference annotation.
[11:10:18] Loading reference annotation.
[11:10:18] Loading reference annotation.
[11:10:18] Loading reference annotation.
[Sat Jan 19 11:10:18 2013] Quantitating transcripts
You are using Cufflinks v2.0.2, which is the most recent release.
Command line:
cufflinks -o merge_IR_Mh/ -F 0.05 -g Mh.gff -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 1 merge_IR_Mh/tmp/mergeSam_fileBL9IlN
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File merge_IR_Mh/tmp/mergeSam_fileBL9IlN doesn't appear to be a valid BAM file, trying SAM...
Error: sort order of reads in BAMs must be the same
[FAILED]
Error: could not execute cufflinks


===============problem occurred above=================

cuffdiff -o cuffdiff_out_IR_Mt merge_IR_Mt/merged.gtf t1_Mt/accepted_hits.bam t2_Mh/accepted_hits.bam

You are using Cufflinks v2.0.2, which is the most recent release.
Error: sort order of reads in BAMs must be the same


===============problem occurred above=================

In summery,
1. There was no problem in running Bowtie-build and Tophat.
2. While running cufflinks, if I included gff file, it did not work.
Even though I dropped gff file, accepted_bam file also made an error.
However, when I dropped gff file but used sam file instead of bam file, four output files were successfully generated.
3. While funning cuffmerge and cuffdiff, it didn't work as well.
4. I checked GFF and SAM/BAM file.
- They are correctly sorted.
- SAM/BAM file: contig0, contig1, contig2, contig3, contig4, ......
- GFF file: contig0, contig1, contig2, contig4...... (no contig3) -> INCOMPLETE
5. accepted_hits.bam file looks like....
@HD VN:1.0 SO:coordinate
@SQ SN:Mh:0000:MhA1_Contig0 LN:237641
@SQ SN:Mh:0001:MhA1_Contig1 LN:17102
@SQ SN:Mh:0002:MhA1_Contig2 LN:88040
@SQ SN:Mh:0003:MhA1_Contig3 LN:1670
@SQ SN:Mh:0004:MhA1_Contig4 LN:27447
@SQ SN:Mh:0005:MhA1_Contig5 LN:2702
@SQ SN:Mh:0006:MhA1_Contig6 LN:3758
@SQ SN:Mh:0007:MhA1_Contig7 LN:156602
@SQ SN:Mh:0008:MhA1_Contig8 LN:1526
@SQ SN:Mh:0009:MhA1_Contig9 LN:106463
@SQ SN:Mh:0010:MhA1_Contig10 LN:7780
@SQ SN:Mh:0011:MhA1_Contig11 LN:67162
@SQ SN:Mh:0012:MhA1_Contig12 LN:16878
@SQ SN:Mh:0013:MhA1_Contig13 LN:73699
@SQ SN:Mh:0014:MhA1_Contig14 LN:78027
6. gff file looks like...
Mh:0000:MhA1_Contig0 Freeze3 mRNA 4027 5970 + ID=MhA1_Contig0.frz3.fgene1;Name=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 4027 4090 15.97 + 0 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 4141 4251 7.12 + 1 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 4311 4390 9.54 + 1 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 4471 4576 11.8 + 0 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 4635 4678 -4.98 + 1 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 4760 4959 14.82 + 0 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 5070 5193 4.65 + 2 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 5243 5274 -3.08 + 0 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 5348 5631 39.94 + 2 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 5747 5841 5.9 + 1 Parent=MhA1_Contig0.frz3.fgene1
Mh:0000:MhA1_Contig0 Freeze3 CDS 5881 5970 9.61 + 0 Parent=MhA1_Contig0.frz3.fgene1

Would you please give me some tips about what I have to fix?

Last edited by syintel87; 01-19-2013 at 07:28 AM.
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