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Forum: Bioinformatics 08-12-2013, 07:15 AM
Replies: 4
Views: 850
Posted By Nicolas
not necessary. Cufflinks do some magic with the...

not necessary. Cufflinks do some magic with the reads leading to FPKM and it's hard to follow precisely all the normalisation step (some people may disagree). I would imagine that the results will be...
Forum: Bioinformatics 08-12-2013, 06:50 AM
Replies: 4
Views: 850
Posted By Nicolas
Do NOT merge replicates! You're losing one useful...

Do NOT merge replicates! You're losing one useful piece of information (ie that they're not the same experiment, they are replicates!)
Instead, use cufflinks and indicates the two BAM files as...
Forum: Bioinformatics 12-04-2012, 11:49 AM
Replies: 14
Views: 5,752
Posted By Nicolas
I'd love to know how many people replied...

I'd love to know how many people replied Cufflinks for quantif and DESeq or edgeR for DE analysis...
Forum: Bioinformatics 11-28-2012, 04:39 PM
Replies: 14
Views: 5,752
Posted By Nicolas
Looking forward to seeing the results. It may...

Looking forward to seeing the results.
It may be too late, but what would also be valuable would be to survey the source of annotations people are using (RefSeq, Gencode, and so on). There are good...
Forum: Bioinformatics 04-26-2012, 07:59 AM
Replies: 8
Views: 2,699
Posted By Nicolas
Hi, I had good results (ie. found the fusion...

Hi,

I had good results (ie. found the fusion known to be present in a cell line) with chimerascan: http://code.google.com/p/chimerascan/

For me, the main advantage on the other softwares is...
Forum: Bioinformatics 04-12-2012, 12:45 PM
Replies: 25
Views: 11,709
Posted By Nicolas
Same here. Tophat2 hangs forever at the same step...

Same here. Tophat2 hangs forever at the same step "Searching for junctions via segment mapping", with paired-end HiSeq fastq reads (~140M reads), with or without --fusion-search option.
I ran it...
Forum: RNA Sequencing 04-10-2012, 10:55 AM
Replies: 37
Views: 18,133
Posted By Nicolas
Hi, It would work the same if your...

Hi,
It would work the same if your File.mapped.bam has been aligned to a transcriptome index.

By the way, I have a general question related to this -r parameter:
Did you guys tried to omit this...
Forum: Bioinformatics 03-30-2012, 12:16 PM
Replies: 3
Views: 2,059
Posted By Nicolas
The authors explained on their website that they...

The authors explained on their website that they introduced the CIGAR F, for fusion, but the consequence is that it breaks the possibility to convert into BAM....
Forum: Bioinformatics 03-08-2012, 07:00 AM
Replies: 21
Views: 8,944
Posted By Nicolas
I think first of all, it means that microRNA and...

I think first of all, it means that microRNA and smallRNA cannot be studied by in canonical RNA-Seq experiment, aimed at studying "longer" RNA (mRNA, lincRNA).
For smallRNA, you do not need to...
Forum: Bioinformatics 02-29-2012, 09:21 AM
Replies: 4
Views: 1,603
Posted By Nicolas
The protocol for smallRNA-Seq is not the same as...

The protocol for smallRNA-Seq is not the same as for "classical" RNA-Seq, with the aim of purifying the sample from long RNA and enriching for "smallRNA". At one point, we do run a gel and select for...
Forum: Bioinformatics 02-29-2012, 09:11 AM
Replies: 4
Views: 1,603
Posted By Nicolas
Hi, Did you collapse identical sequences...

Hi,

Did you collapse identical sequences (you should not...)? How did you size-selected your RNA? Which tissues is it from (piRNA are abundant in the germline)?
Forum: Bioinformatics 02-22-2012, 11:19 AM
Replies: 12
Views: 4,053
Posted By Nicolas
Thanks a lot. I am really looking forward for...

Thanks a lot.
I am really looking forward for Tophat2 ;-)
Forum: Bioinformatics 02-22-2012, 09:37 AM
Replies: 30
Views: 10,747
Posted By Nicolas
Hi, I am sure Tophat-fusion is awesome, but it...

Hi,
I am sure Tophat-fusion is awesome, but it would be great to get some feedback on the problems observed by several users and reported here :
...
Forum: Bioinformatics 02-22-2012, 09:35 AM
Replies: 12
Views: 4,053
Posted By Nicolas
Same here. Systematic crash at the last step of...

Same here. Systematic crash at the last step of tophat-fusion (tophat-reports).
The sample comes from an HiSeq2000 and the fastq files are 21Go (compressed. ~140M reads)
The 4 files it uses are...
Forum: Bioinformatics 02-15-2012, 07:50 AM
Replies: 5
Views: 3,930
Posted By Nicolas
Hi, Which mode did you use Cufflinks with?...

Hi,

Which mode did you use Cufflinks with? with a reference file, in RABT mode, or de-novo?
Forum: RNA Sequencing 02-11-2012, 02:42 PM
Replies: 37
Views: 18,133
Posted By Nicolas
Oups, my bad. Field 9 is actually the observed...

Oups, my bad. Field 9 is actually the observed template length, and therefore, in your case, the inner-distance is negative... I never had to run Tophat with a negative inner-distance, but you should...
Forum: RNA Sequencing 02-11-2012, 10:02 AM
Replies: 37
Views: 18,133
Posted By Nicolas
Sure. The point here is to map to the genome to...

Sure. The point here is to map to the genome to estimate the size of the insert (from full length alignments), and feed this parameter into Tophat, to map to the transcriptome (or at least, to allow...
Forum: RNA Sequencing 02-11-2012, 10:00 AM
Replies: 37
Views: 18,133
Posted By Nicolas
Hi, Using the command-line above will...

Hi,

Using the command-line above will summarize the values in field 9 ($9), which, in the SAM format indicates the distance between the "left" position of each reads in the pair. So, the inner...
Forum: Bioinformatics 02-09-2012, 07:08 AM
Replies: 6
Views: 1,737
Posted By Nicolas
Does your multifasta file contains one entry per...

Does your multifasta file contains one entry per gene?
If so, it should be easy to count the number of reads mapping to each entry (samtools idxstats <aln.bam> for instance). You can then normalize...
Forum: Bioinformatics 02-08-2012, 02:13 PM
Replies: 6
Views: 1,737
Posted By Nicolas
Could you post the command you're using? Which...

Could you post the command you're using?
Which Cufflinks mode are you using, de novo (default), with a reference annotation (-G) or RABT (-g)?
Is there a complete coverage of your gene? If not...
Forum: Bioinformatics 02-08-2012, 01:56 PM
Replies: 6
Views: 1,737
Posted By Nicolas
Could it be multiple isoforms of the same gene?

Could it be multiple isoforms of the same gene?
Forum: Bioinformatics 02-07-2012, 11:34 AM
Replies: 21
Views: 8,944
Posted By Nicolas
That does not make sense to me. Unless it is an...

That does not make sense to me. Unless it is an option in either Cufflinks or Cuffdiff, but I have never saw a log relationship between Cufflinks and Cuffdiff outputs.

Honey, how did you run...
Forum: Bioinformatics 01-25-2012, 07:28 AM
Replies: 7
Views: 3,848
Posted By Nicolas
fastq-groomer does not remove the 3'end adapter...

fastq-groomer does not remove the 3'end adapter sequence present on the 3'end of your sequences (correct me if I am wrong).

What is the size of the sequences you tried to align?

If they are...
Forum: Bioinformatics 01-24-2012, 02:03 PM
Replies: 7
Views: 3,848
Posted By Nicolas
You can use Bowtie (or BWA, or any short-read...

You can use Bowtie (or BWA, or any short-read aligner). Create an index from the hairpin.fa file (bowtie-build) and map your fastq directly to the index. Specify the number of mismatches you want (up...
Forum: Bioinformatics 01-19-2012, 08:00 AM
Replies: 24
Views: 14,335
Posted By Nicolas
I think that is a novelty of Tophat 1.4. You...

I think that is a novelty of Tophat 1.4.
You don't need to provide the file "Homo_sapiens.GRCh37.65.fa" but it is generated from Genome Index (in your case:...
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