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Forum: RNA Sequencing 01-28-2015, 07:49 AM
Replies: 0
Views: 1,992
Posted By Julien Roux
Influence of quality of genome assembly for RNA-seq read mapping

Dear all,
I am wondering how much the quality of the reference genome assembly influences RNA-seq read mapping (for example Illumina reads mapped to the genome with Tophat). I could not find any...
Forum: Bioinformatics 06-10-2013, 11:28 AM
Replies: 648
Views: 189,812
Posted By Julien Roux
Solved: alignment on one strand only

Just a quick message to let users know that my problem is solved and was due to an error when generating the Bowtie index files for one of the strands. All index files were present, but one of them...
Forum: Bioinformatics 06-08-2013, 08:27 PM
Replies: 648
Views: 189,812
Posted By Julien Roux
Hi Felix Actually I was running bismark on a...

Hi Felix
Actually I was running bismark on a cluster and I requested 32g of memory. I have now run it with 48g and I'm still encountering the same issue...
Do you have any idea of what else could...
Forum: Bioinformatics 06-08-2013, 07:17 AM
Replies: 648
Views: 189,812
Posted By Julien Roux
Thanks Felix for your answer. I am now...

Thanks Felix for your answer.

I am now facing another problem: when I run bismark of some of my samples, reads end up mapped to only the top or the bottom strand... This is not happening for all...
Forum: Bioinformatics 05-30-2013, 11:47 AM
Replies: 648
Views: 189,812
Posted By Julien Roux
Dear Felix, I am wondering if Bismark can be...

Dear Felix,
I am wondering if Bismark can be fed with an input stream in a pipeline.
For example would that work?
cutadapt -b ACTGCTCG input_file.fastq | bismark -n 1 --solexa1.3-quals --bam...
Forum: Bioinformatics 05-17-2013, 09:22 AM
Replies: 38
Views: 27,311
Posted By Julien Roux
Here is an answer to billstevens (many questions...

Here is an answer to billstevens (many questions got intertwined):
It seems that you've already launched the tophat command (unsuccessfully) before, which led to the creation of an...
Forum: Bioinformatics 04-12-2013, 01:07 PM
Replies: 5
Views: 5,686
Posted By Julien Roux
I am in the process of writing a perl script that...

I am in the process of writing a perl script that generates the file junctions.bed from a given tophat alignment BAM file and I discovered a small bug that sometimes affects the definition of the...
Forum: Bioinformatics 04-10-2013, 02:26 PM
Replies: 5
Views: 5,686
Posted By Julien Roux
Tophat: generate junctions.bed file from BAM file

Dear all,
I have previously mapped RNA-seq samples to the human genome using Tophat 2.0.6. This generated an "accepted_hits.bam" result file, and a "junctions.bed" file I am interested in for...
Forum: RNA Sequencing 03-26-2013, 08:32 AM
Replies: 1
Views: 3,673
Posted By Julien Roux
Tophat discovers junctions at same coordinates on opposite strands

Dear all,

I have a question regarding junctions discovery in Tophat (version 2.0.6). I have mapped an unstranded RNA-seq library (single-end 107bp reads) to the human genome and provided gene...
Forum: Bioinformatics 01-22-2013, 08:28 AM
Replies: 3
Views: 1,818
Posted By Julien Roux
Thanks Alejandro for you help! Indeed the "TRT"...

Thanks Alejandro for you help!
Indeed the "TRT" fucntions worked a lot more quickly and with a reasonable amount of memory.
When you say:

... I actually find that the p-values are well...
Forum: Bioinformatics 01-08-2013, 02:03 PM
Replies: 3
Views: 1,818
Posted By Julien Roux
DEXSeq memory requirements

Dear all,
I am analyzing a RNA-seq experiment with DEXSeq. There are 73 samples (from 23 different conditions).
When trying to estimate the dispersions for the whole exonCountSet (all conditions...
Forum: Bioinformatics 11-27-2012, 01:30 PM
Replies: 3
Views: 3,023
Posted By Julien Roux
Hi Simon, Thanks for your answer. However I am...

Hi Simon,
Thanks for your answer. However I am not fully convinced that it is not an issue...

In my dataset at least 40% of the reads overlap an exon junction. If you count these read twice when...
Forum: Bioinformatics 11-15-2012, 03:19 PM
Replies: 3
Views: 3,023
Posted By Julien Roux
DEXSeq gene level counts

Dear DEXSeq authors,
I have a question related to the following paragraph in the DEXSeq vignette:


I am wondering if this problem is also affecting the tests for differential expression of exons...
Forum: Illumina/Solexa 07-19-2012, 10:11 AM
Replies: 7
Views: 3,152
Posted By Julien Roux
Mmmm, I'm getting confused... Now I understand...

Mmmm, I'm getting confused...
Now I understand you don't agree with Simon and what was discussed here: https://mailman.stat.ethz.ch/pipermail/bioconductor/2012-March/044094.html
(Basically, it is...
Forum: Illumina/Solexa 07-19-2012, 08:07 AM
Replies: 7
Views: 3,152
Posted By Julien Roux
Arrow Thanks again Alejandro, If I understood...

Thanks again Alejandro,
If I understood correctly, you suggest to:
- estimate the dispersions on the whole ecs using "estimateDispersions"
- fit the coefficients of the dispersion function from...
Forum: Illumina/Solexa 07-13-2012, 09:57 AM
Replies: 7
Views: 3,152
Posted By Julien Roux
Thanks Alejandro, Yes indeed, that's what I...

Thanks Alejandro,
Yes indeed, that's what I ended up doing:


> allExonsPairwise <- newExonCountSet(countData = counts(allExons)[, -c(1:3)], design = design(allExons)[-c(1:3),], geneIDs =...
Forum: Illumina/Solexa 07-12-2012, 08:49 AM
Replies: 7
Views: 3,152
Posted By Julien Roux
Pairwise comparisons in DEXSeq

Dear all,
I am analyzing a RNA-seq experiment including more than 2 conditions. As discussed in this topic, I have created a global exonCountSet, including all conditions and their replicates, to...
Forum: Bioinformatics 04-12-2012, 09:01 AM
Replies: 4
Views: 3,165
Posted By Julien Roux
Thanks Geo for your clarifications that the -g 1...

Thanks Geo for your clarifications that the -g 1 option is eventually applied to all the reads. I guess that if a user wants more tuning on the transcriptome mapping step, it might be preferable to...
Forum: Bioinformatics 04-11-2012, 07:59 AM
Replies: 4
Views: 3,165
Posted By Julien Roux
Yes indeed I think this is what happens: I have...

Yes indeed I think this is what happens: I have just checked a run output with -x 1 and the mapping rate is only 16%. Most reads are thrown away because they map multiple locations.



I thought...
Forum: Bioinformatics 04-10-2012, 10:02 AM
Replies: 4
Views: 3,165
Posted By Julien Roux
Tophat 1.4.1 in -G mode

Dear all,

I am trying to map reads from an Illumina RNA-seq run to the human genome.
I am using the command:
~/bin/tophat-1.4.1.Linux_x86_64/tophat --solexa1.3-quals --library-type fr-unstranded...
Forum: RNA Sequencing 04-06-2012, 07:35 AM
Replies: 6
Views: 4,520
Posted By Julien Roux
Probably this is part of the problem since by...

Probably this is part of the problem since by default Tophat only allows 2 mismatches on the whole read. I had a similar problem when analyzing reads of 107bp. Switching to --bowtie-n mode might help...
Forum: Illumina/Solexa 02-16-2012, 08:27 AM
Replies: 5
Views: 5,745
Posted By Julien Roux
Actually not. Some high concentration libraries...

Actually not. Some high concentration libraries also show a noisy peak, while some low concentration libraries look perfectly fine.
Did you sequence the low concentration libraries you're mentioning?
Forum: Illumina/Solexa 02-15-2012, 01:38 PM
Replies: 5
Views: 5,745
Posted By Julien Roux
NextGenSeq: Yes, the DNA is fragmented before...

NextGenSeq: Yes, the DNA is fragmented before cDNA synthesis, with the Elute, Prime, Fragment Mix (EPF) provided by Illumina. I'm not sure the problem comes from this step since other libraries of...
Forum: Illumina/Solexa 02-15-2012, 11:02 AM
Replies: 5
Views: 5,745
Posted By Julien Roux
weird noisy peaks on bioanalyzer

Hi everyone,
We are getting some weird results when running RNA-seq libraries on Agilent Bioanalyzer.

The libraries show a peak at the expected length, but the peak itself is very noisy, with a...
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