Originally posted by greggrant
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Originally posted by Cole Trapnell View PostWait, that's only one read. Are you aligning all of them that way, individually on the command line?
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454 support?
Trimming all of the sequences to the same length seems like a bad idea. Any idea when tophat / bowtie will support 454 variable length reads?
Greg -- how did you take your mouse reads and get them to be a uniform length? Straight trimming? Segmenting?
Thanks,
Anand
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Bowtie already supports variable length reads. It's TopHat that has a problem with them. Fixing TopHat for 454 would require more work than I have time to do in the near future, as I am focused primarily on Cufflinks (and graduating). It's on the list of things to do, but so are many other things...
One obstacle to me making the changes is that I don't currently have any 454 RNA-Seq data to work with, as none of my collaborators use it. If someone was willing to provide me with a small test set (perhaps a chromosome's worth), I could at least assess how much work it will actually be to add support for this. I'd keep it confidential, of course.
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how much data?
Cole,
How much data is a "chromosome's worth"? (I ask only because some chromosomes are huge, and others not so huge).
I've got 9 reads, 312-385 average read length, 110-170 mbases per read.
How much of that would you like? Fastq? SFF?
Thanks,
Anand
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Tophat Coverage Plots
I have a question regarding the coverage files output by tophat. What exactly is required for a read to be include in a coverage map? If Tophat breaks up a read into four pieces and only one maps, is that piece included? Also, how are reads that map to multiple locations handled? Any information is greatly appreciated, thank you.
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Originally posted by greggrant View PostI have a question regarding the coverage files output by tophat. What exactly is required for a read to be include in a coverage map? If Tophat breaks up a read into four pieces and only one maps, is that piece included? Also, how are reads that map to multiple locations handled? Any information is greatly appreciated, thank you.
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Originally posted by acpatel View PostTrimming all of the sequences to the same length seems like a bad idea. Any idea when tophat / bowtie will support 454 variable length reads?
Greg -- how did you take your mouse reads and get them to be a uniform length? Straight trimming? Segmenting?
Thanks,
Anand
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Hi All,
I am getting this error message on Mac OS X 10.5.8
Could you please help me what did I install wrong?
Thanks in advance!
tophat -r 20 test_ref reads_1.fq reads_2.fq
[2012-10-22 16:52:02] Beginning TopHat run (v2.0.5)
-----------------------------------------------
[2012-10-22 16:52:02] Checking for Bowtie
Bowtie version: 2.0.0.7
[2012-10-22 16:52:02] Checking for Samtools
Samtools version: 0.1.18.0
[2012-10-22 16:52:02] Checking for Bowtie index files
[2012-10-22 16:52:02] Checking for reference FASTA file
[2012-10-22 16:52:02] Generating SAM header for test_ref
format: fastq
quality scale: phred33 (default)
[2012-10-22 16:52:02] Preparing reads
[FAILED]
Error running 'prep_reads'
dyld: unknown required load command 0x80000022
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HI.. I am a new user of tophat and I have illumina generated RNA seq single end reads of 35ntd length. Here is the error I am getting when I tried to run tophat...
root@ubuntu:~# tophat -g 1 -p 4 -o '/media/bv/My Passport/output' '/home/bv/Desktop/b/bowtie2-2.1.0/index/hg19' '/media/bv/My Passport/Normal/normal.fastq'
[2013-07-02 12:46:03] Beginning TopHat run (v2.0.3)
-----------------------------------------------
[2013-07-02 12:46:03] Checking for Bowtie
Bowtie version: 2.0.0.6
[2013-07-02 12:46:03] Checking for Samtools
Samtools version: 0.1.19.0
[2013-07-02 12:46:03] Checking for Bowtie index files
[2013-07-02 12:46:03] Checking for reference FASTA file
Warning: Could not find FASTA file /home/bv/Desktop/b/bowtie2-2.1.0/index/hg19.fa
[2013-07-02 12:46:03] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/bin/bowtie2-inspect /home/bv/Desktop/b/bowtie2-2.1.0/index/hg19 > /media/bv/My Passport/output/tmp/hg19.fa
[2013-07-02 12:51:29] Generating SAM header for /home/bv/Desktop/b/bowtie2-2.1.0/index/hg19
format: fastq
quality scale: phred33 (default)
[2013-07-02 12:51:40] Preparing reads
left reads: min. length=35, max. length=35, 15669232 kept reads (5155 discarded)
Warning: you have only one segment per read.
If the read length is greater than or equal to 45bp,
we strongly recommend that you decrease --segment-length to about half the read length because TopHat will work better with multiple segments
open: No such file or directory
[main_samview] fail to open "/media/bv/My" for reading.
Warning: junction database is empty!
open: No such file or directory
[main_samview] fail to open "/media/bv/My" for reading.
[2013-07-02 12:54:33] Reporting output tracks
[FAILED]
Error running /usr/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /media/bv/My Passport/output/ --max-multihits 1 --max-seg-multihits 10 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --max-mismatches 2 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p4 --no-closure-search --no-microexon-search --sam-header /media/bv/My Passport/output/tmp/hg19_genome.bwt.samheader.sam --samtools=/usr/local/bin/samtools --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 /media/bv/My Passport/output/tmp/hg19.fa /media/bv/My Passport/output/junctions.bed /media/bv/My Passport/output/insertions.bed /media/bv/My Passport/output/deletions.bed /media/bv/My Passport/output/fusions.out /media/bv/My Passport/output/tmp/accepted_hits /media/bv/My Passport/output/tmp/left_kept_reads.bam
Loaded 0 junctions
please help me in learning what has went wrong..
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Hi Next
I think you should know what is your read length and insert size. Tophat takes 250 insert size by default.
Now my question is how can we change the default option ?
may someone answer my question posted here: http://seqanswers.com/forums/showthr...098#post112098
Originally posted by Nextgenanalysis View PostHI.. I am a new user of tophat and I have illumina generated RNA seq single end reads of 35ntd length. Here is the error I am getting when I tried to run tophat...
root@ubuntu:~# tophat -g 1 -p 4 -o '/media/bv/My Passport/output' '/home/bv/Desktop/b/bowtie2-2.1.0/index/hg19' '/media/bv/My Passport/Normal/normal.fastq'
[2013-07-02 12:46:03] Beginning TopHat run (v2.0.3)
-----------------------------------------------
[2013-07-02 12:46:03] Checking for Bowtie
Bowtie version: 2.0.0.6
[2013-07-02 12:46:03] Checking for Samtools
Samtools version: 0.1.19.0
[2013-07-02 12:46:03] Checking for Bowtie index files
[2013-07-02 12:46:03] Checking for reference FASTA file
Warning: Could not find FASTA file /home/bv/Desktop/b/bowtie2-2.1.0/index/hg19.fa
[2013-07-02 12:46:03] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/bin/bowtie2-inspect /home/bv/Desktop/b/bowtie2-2.1.0/index/hg19 > /media/bv/My Passport/output/tmp/hg19.fa
[2013-07-02 12:51:29] Generating SAM header for /home/bv/Desktop/b/bowtie2-2.1.0/index/hg19
format: fastq
quality scale: phred33 (default)
[2013-07-02 12:51:40] Preparing reads
left reads: min. length=35, max. length=35, 15669232 kept reads (5155 discarded)
Warning: you have only one segment per read.
If the read length is greater than or equal to 45bp,
we strongly recommend that you decrease --segment-length to about half the read length because TopHat will work better with multiple segments
open: No such file or directory
[main_samview] fail to open "/media/bv/My" for reading.
Warning: junction database is empty!
open: No such file or directory
[main_samview] fail to open "/media/bv/My" for reading.
[2013-07-02 12:54:33] Reporting output tracks
[FAILED]
Error running /usr/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /media/bv/My Passport/output/ --max-multihits 1 --max-seg-multihits 10 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --max-mismatches 2 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p4 --no-closure-search --no-microexon-search --sam-header /media/bv/My Passport/output/tmp/hg19_genome.bwt.samheader.sam --samtools=/usr/local/bin/samtools --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 /media/bv/My Passport/output/tmp/hg19.fa /media/bv/My Passport/output/junctions.bed /media/bv/My Passport/output/insertions.bed /media/bv/My Passport/output/deletions.bed /media/bv/My Passport/output/fusions.out /media/bv/My Passport/output/tmp/accepted_hits /media/bv/My Passport/output/tmp/left_kept_reads.bam
Loaded 0 junctions
please help me in learning what has went wrong..
Comment
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Originally posted by Nextgenanalysis View PostHI.. I am a new user of tophat and I have illumina generated RNA seq single end reads of 35ntd length. Here is the error I am getting when I tried to run tophat...
root@ubuntu:~# tophat -g 1 -p 4 -o '/media/bv/My Passport/output' '/home/bv/Desktop/b/bowtie2-2.1.0/index/hg19' '/media/bv/My Passport/Normal/normal.fastq'
(... much deleted ...)
[main_samview] fail to open "/media/bv/My" for reading.
(... more deleted ...)
please help me in learning what has went wrong..
BTW, jp's comment has nothing to do with your problem.
Comment
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