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Forum: Sample Prep / Library Generation 11-19-2014, 12:36 AM
Replies: 3
Views: 3,363
Posted By blanco
Hi, all the posts here on DSN are rather old...

Hi,
all the posts here on DSN are rather old and likewise publication using the method are few and date a couple of years back.

Thus, I was wondering if people are still using DSN normalization...
Forum: Sample Prep / Library Generation 08-17-2014, 11:55 PM
Replies: 0
Views: 954
Posted By blanco
Small RNA extractions from plasma

Hi all who have some experience/knowledge in working with RNA from plasma.

I just did some extractions and was deterred when I ran the RNA Nano on the bioanalyzer and found no trace of RNA.
I...
Forum: Sample Prep / Library Generation 08-11-2014, 05:27 AM
Replies: 8
Views: 5,017
Posted By blanco
Hi all who have some experience/knowledge in...

Hi all who have some experience/knowledge in working with RNA from plasma.

I just did some extractions and was deterred when I ran the RNA Nano on the bioanalyzer and found no trace of RNA.
I...
Forum: Bioinformatics 02-25-2014, 03:35 AM
Replies: 15
Views: 3,793
Posted By blanco
Hi Federico, have you confirmed that the data...

Hi Federico,
have you confirmed that the data you have is indeed strand specific?
You can use the infer_experiment.py script from the RSeQC package to check it out -...
Forum: Bioinformatics 08-30-2013, 06:03 AM
Replies: 9
Views: 5,715
Posted By blanco
I ran into similar problem: tophat generated all...

I ran into similar problem: tophat generated all files except accepted_hits.bam.
I made some more disk space available and then it ran fine.
Forum: Bioinformatics 06-09-2013, 03:26 AM
Replies: 3
Views: 1,428
Posted By blanco
RSeQC: https://code.google.com/p/rseqc/

RSeQC:
https://code.google.com/p/rseqc/
Forum: Bioinformatics 04-05-2013, 07:09 AM
Replies: 19
Views: 5,860
Posted By blanco
I would also be interested in a clear answer to...

I would also be interested in a clear answer to this question.

My worry is that by supplying tophat with an annotation file if it is then biased toward aligning to genes in the annotation instead...
Forum: Bioinformatics 01-17-2013, 05:44 AM
Replies: 136
Views: 41,421
Posted By blanco
Thanks again for your quick reply fkrueger. ...

Thanks again for your quick reply fkrueger.

What, however, if I do not want to use the default 13bp adapter but instead want to use the whole 63 bp index-adapter (index = red):...
Forum: Bioinformatics 01-17-2013, 03:57 AM
Replies: 14
Views: 14,632
Posted By blanco
Thanks for your quick reply fkrueger - this looks...

Thanks for your quick reply fkrueger - this looks to be something really useful. I have already asked one question in the appropriate thread:...
Forum: Bioinformatics 01-17-2013, 03:56 AM
Replies: 136
Views: 41,421
Posted By blanco
Hi, I am wondering about the -a and -a2...

Hi, I am wondering about the -a and -a2 parameters for paired end reads

For example my libraries were made using the Illumina TruSeq adapters, one of which is:...
Forum: Bioinformatics 01-17-2013, 02:17 AM
Replies: 14
Views: 14,632
Posted By blanco
I am also interested to know the answer to some...

I am also interested to know the answer to some of these questions.

Perhaps to put it more simply: When trimming paired end reads, should the cutadapt command be exactly the same for both...
Forum: RNA Sequencing 10-18-2012, 04:23 AM
Replies: 23
Views: 66,280
Posted By blanco
Hi folks - hope some of you can help me clarify...

Hi folks - hope some of you can help me clarify something about adapter contamination and adapter trimming.

I made TruSeq Illumina libraries and sequenced them for 100bp paired end reads.

When...
Forum: Bioinformatics 10-13-2012, 02:07 AM
Replies: 8
Views: 9,613
Posted By blanco
Hi masterpiece, this is the same pattern I get...

Hi masterpiece,
this is the same pattern I get for the dUTP method and I use fr-fristrand in tophat so I assume that would also apply to your reads.

This is impressive strand specificity, I...
Forum: Bioinformatics 10-11-2012, 02:18 AM
Replies: 2
Views: 1,355
Posted By blanco
Hi, I suppose there could be a couple of...

Hi,
I suppose there could be a couple of reasons for this. It could be two different transcripts from the same gene. Or it could be antisense transcription.

If you look at the column...
Forum: RNA Sequencing 10-10-2012, 12:07 PM
Replies: 2
Views: 3,846
Posted By blanco
Hi oomvel, padj means adjusted p-value in the...

Hi oomvel,
padj means adjusted p-value in the sense it is adjusted for multiple testing.

I am perhaps not the most experienced user here to answer this but, when you do a lot of statistical tests...
Forum: Bioinformatics 10-10-2012, 06:58 AM
Replies: 0
Views: 1,730
Posted By blanco
Unplaced chromosomes / supercontigs during mapping

Hi all,
when you download the human reference genome it usually includes many different kinds of unplaced supercontigs such as GL000217, GL000218, etc.

I am wondering whether they should be...
Forum: Bioinformatics 10-10-2012, 01:55 AM
Replies: 5
Views: 17,797
Posted By blanco
Hi Liye, I believe this is caused by fpkm...

Hi Liye,
I believe this is caused by fpkm values of zero. When the program takes the log of zero it becomes minus infinite (-Inf) and these values are excluded from the analysis.

I think there...
Forum: RNA Sequencing 10-08-2012, 12:02 AM
Replies: 1
Views: 1,086
Posted By blanco
Hi, I have a similar problem. I ran cuffdiff...

Hi,
I have a similar problem. I ran cuffdiff and all output files seem ok except the cds ones, which are empty.

I ran cuffdiff with the -u option for the merged .gtf file from cuffmerge as well...
Forum: Bioinformatics 09-19-2012, 02:43 AM
Replies: 1
Views: 1,927
Posted By blanco
It is not unusual (I think) that many transcripts...

It is not unusual (I think) that many transcripts have an FPKM of value 0 - just means that that transcript is not expressed.

However if all transcripts have an FPKM value of 0 then something is...
Forum: Bioinformatics 08-15-2012, 07:04 AM
Replies: 6
Views: 5,813
Posted By blanco
I also have a problem with ReorderSam. The...

I also have a problem with ReorderSam.
The error I get is: New reference sequence does not contain a matching contig for chr1.

Any help/input appreciated.
Forum: Bioinformatics 05-30-2012, 05:04 AM
Replies: 5
Views: 1,449
Posted By blanco
Hi folks, I used Picard to remove duplicates...

Hi folks,
I used Picard to remove duplicates but it removed around 60% of the reads (samtolls rmdup removed even more). That seems quite a lot - I have heard around 30% is normal.

My library was...
Forum: Bioinformatics 05-30-2012, 04:46 AM
Replies: 1
Views: 2,072
Posted By blanco
You probably have figured something out by now...

You probably have figured something out by now but still:
tophat now has a '-library--type' option which can tell you from which strand the read came from (assuming the library is strand specific).
Forum: Bioinformatics 05-30-2012, 04:42 AM
Replies: 8
Views: 9,613
Posted By blanco
Hi, I used tophat to map dUTP strand specific...

Hi,
I used tophat to map dUTP strand specific libraries and I used '–library–-type fr-firststrand'.

There is a nice quality control package called RSeQC...
Forum: Bioinformatics 05-15-2012, 08:12 AM
Replies: 2
Views: 2,337
Posted By blanco
Quantifying antisense transcription from strand specific data

Hi all,
I have sequences from strand specific libraries made with the 'dUTP second strand marking' protocol.

I was wondering what would be the best way to quantify antisense transcription of the...
Forum: Bioinformatics 05-10-2012, 05:29 AM
Replies: 1
Views: 1,850
Posted By blanco
After some research and correspondence with folks...

After some research and correspondence with folks from the community I have confirmed that the XS:A tag indeed infers from which strand the read comes from.

Just in case some people are wondering...
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