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Hello members
I have downloaded all my genome sequence indexes and GTF transcript annotation files from igenomes
http://cufflinks.cbcb.umd.edu/igenomes.html, namely
ftp://igenome:[email protected]..._GRCh37.tar.gz
So that everything is supposed to be perfect. Indeed, tophat and cufflinks ran according to manual without any problems, but cuffmerge is giving me
errors.
Cuffmerge command I use is:
cufflinks-2.2.1/cuffmerge --num-threads 4 -o TumorVsNorm -s ./Ensembl/GRCh37/Sequence/Bowtie2Index/genome.fa -g ./Ensembl/GRCh37/Annotation/Archives/archive-current/Genes/genes.gtf TumorVsNorm_assembly.txt
Error is
Preparing output location /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/
[Wed Sep 24 16:55:52 2014] Converting GTF files to SAM
[16:55:52] Loading reference annotation.
[16:56:09] Loading reference annotation.
[16:56:28] Loading reference annotation.
[16:56:42] Loading reference annotation.
[16:56:58] Loading reference annotation.
[16:57:12] Loading reference annotation.
[16:57:28] Loading reference annotation.
[16:57:42] Loading reference annotation.
[Wed Sep 24 16:58:02 2014] Quantitating transcripts
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
Command line:
cufflinks -o /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/ -F 0.05 -g /home/makarovv/RnaSeq/tuxedo/bowtie2-2.2.3/reference/b37/Homo_sapiens/Ensembl/GRCh37/Annotation/Archives/archive-current/Genes/genes.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 4 /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/tmp/mergeSam_file7apIHH
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/tmp/mergeSam_file7apIHH doesn't appear to be a valid BAM file, trying SAM...
[16:58:02] Loading reference annotation.
[16:58:36] Inspecting reads and determining fragment length distribution.
Error: this SAM file doesn't appear to be correctly sorted!
current hit is at GL000199.1:1175, last one was at GL000197.1:16397
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.
[FAILED]
Error: could not execute cufflinks
Many thanks in advance
Vlad
I have downloaded all my genome sequence indexes and GTF transcript annotation files from igenomes
http://cufflinks.cbcb.umd.edu/igenomes.html, namely
ftp://igenome:[email protected]..._GRCh37.tar.gz
So that everything is supposed to be perfect. Indeed, tophat and cufflinks ran according to manual without any problems, but cuffmerge is giving me
errors.
Cuffmerge command I use is:
cufflinks-2.2.1/cuffmerge --num-threads 4 -o TumorVsNorm -s ./Ensembl/GRCh37/Sequence/Bowtie2Index/genome.fa -g ./Ensembl/GRCh37/Annotation/Archives/archive-current/Genes/genes.gtf TumorVsNorm_assembly.txt
Error is
Preparing output location /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/
[Wed Sep 24 16:55:52 2014] Converting GTF files to SAM
[16:55:52] Loading reference annotation.
[16:56:09] Loading reference annotation.
[16:56:28] Loading reference annotation.
[16:56:42] Loading reference annotation.
[16:56:58] Loading reference annotation.
[16:57:12] Loading reference annotation.
[16:57:28] Loading reference annotation.
[16:57:42] Loading reference annotation.
[Wed Sep 24 16:58:02 2014] Quantitating transcripts
Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
Command line:
cufflinks -o /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/ -F 0.05 -g /home/makarovv/RnaSeq/tuxedo/bowtie2-2.2.3/reference/b37/Homo_sapiens/Ensembl/GRCh37/Annotation/Archives/archive-current/Genes/genes.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 4 /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/tmp/mergeSam_file7apIHH
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File /ifs/e63data/makarovv/HCC/RNASeq/TumorVsNorm/tmp/mergeSam_file7apIHH doesn't appear to be a valid BAM file, trying SAM...
[16:58:02] Loading reference annotation.
[16:58:36] Inspecting reads and determining fragment length distribution.
Error: this SAM file doesn't appear to be correctly sorted!
current hit is at GL000199.1:1175, last one was at GL000197.1:16397
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.
[FAILED]
Error: could not execute cufflinks
Many thanks in advance
Vlad