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Forum: Illumina/Solexa 03-03-2015, 07:43 AM
Replies: 13
Views: 2,746
Posted By NGSfan
GenoMax , thanks a lot for bringing up the ease...

GenoMax , thanks a lot for bringing up the ease of using dual barcodes. I will bring this up with the wetlab. I think in the end, this is the real solution. Getting them to adapt to using dual...
Forum: Illumina/Solexa 03-03-2015, 12:28 AM
Replies: 13
Views: 2,746
Posted By NGSfan
We are running single end reads 50 bp + 7bp...

We are running single end reads 50 bp + 7bp index. I had thought that dual indexes were only possible with paired end reads?

Maybe I'm wrong, I just had a look at this Illumina manual:
...
Forum: Illumina/Solexa 03-02-2015, 02:17 PM
Replies: 13
Views: 2,746
Posted By NGSfan
Thanks a lot Tom for pointing this article to me...

Thanks a lot Tom for pointing this article to me - that indeed explains a lot.

I think these are all good suggestions, and I will relay them to the wetlab team. In practice though, I suspect it...
Forum: Illumina/Solexa 03-02-2015, 01:26 PM
Replies: 13
Views: 2,746
Posted By NGSfan
Brian, thanks a lot for offering to try your...

Brian, thanks a lot for offering to try your tool! I will take a shot at it.

The example you give would help me to figure out species contamination and that would be very useful in my scenario.
...
Forum: Illumina/Solexa 02-28-2015, 08:58 AM
Replies: 13
Views: 2,746
Posted By NGSfan
QC approaches for catching multiplexed libraries cross contamination (via adapters)?

Dear fellow Illumina users,

Our wetlab people occassionally cross contaminate multiplexed RNA-seq samples in the same lane, most likely at the adapter ligation stage. So for example, in the same...
Forum: Bioinformatics 01-28-2015, 08:35 AM
Replies: 5
Views: 2,298
Posted By NGSfan
I have been using limma for a while now, but I am...

I have been using limma for a while now, but I am also a bit unsure about the syntax of the model.matrix command when it comes to batch effects, random effects, paired design, etc.

If I...
Forum: Bioinformatics 08-02-2013, 03:06 AM
Replies: 12
Views: 4,901
Posted By NGSfan
Thanks for the update jparsons. Interesting that...

Thanks for the update jparsons. Interesting that it switched to NOTEST in the newer version.

Are you using the same Ensembl GTF with Cuffdiff as you do with RSEM?
Forum: Bioinformatics 07-31-2013, 09:25 AM
Replies: 12
Views: 4,901
Posted By NGSfan
Wow.. that one is a tricky case. They are both...

Wow.. that one is a tricky case. They are both transcribed off the minus strand. I looked at it in the genome browser:
...
Forum: Bioinformatics 07-31-2013, 08:08 AM
Replies: 12
Views: 4,901
Posted By NGSfan
Thanks for sharing this! What were the RPKMs of...

Thanks for sharing this! What were the RPKMs of these transcripts?

RNA-seq suffers from random sampling so if the overall gene expression level is low, then the chance is higher that reads will...
Forum: Bioinformatics 07-30-2013, 07:38 AM
Replies: 12
Views: 4,901
Posted By NGSfan
You should read the paper "Computational methods...

You should read the paper "Computational methods for transcriptome annotation and quantification using RNA-seq" in Nature Methods.

In that paper the cuffdiff authors argue why transcript...
Forum: Bioinformatics 05-29-2013, 06:04 AM
Replies: 218
Views: 118,350
Posted By NGSfan
hi priya, you may want to post this and carry on...

hi priya, you may want to post this and carry on the conversation at the google groups for rna-star:

https://groups.google.com/forum/#!forum/rna-star
Forum: RNA Sequencing 04-16-2013, 07:10 AM
Replies: 4
Views: 4,098
Posted By NGSfan
If my interpretation or understanding of the...

If my interpretation or understanding of the --max-frag-multihits 1 option is correct, this tells cuffdiff to limit itself to using reads that align uniquely to one locus, and to ignore reads that...
Forum: RNA Sequencing 04-16-2013, 05:22 AM
Replies: 1
Views: 2,207
Posted By NGSfan
Please find my solution in this thread...

Please find my solution in this thread http://seqanswers.com/forums/showthread.php?t=26041. The problem is that the default settings between cufflinks and cuffdiff are different.



btw- there is...
Forum: Bioinformatics 03-26-2013, 06:19 AM
Replies: 14
Views: 3,805
Posted By NGSfan
While I don't like re-inventing the wheel, I'm...

While I don't like re-inventing the wheel, I'm not a fan of BioPerl - a lot of overhead and dependencies to do simple tasks like reading in FASTQ files.
Forum: Bioinformatics 02-22-2013, 08:27 AM
Replies: 9
Views: 10,301
Posted By NGSfan
If you input a GTF file into STAR you can have it...

If you input a GTF file into STAR you can have it index the known splice junctions for you...
Forum: Bioinformatics 02-22-2013, 08:25 AM
Replies: 9
Views: 10,301
Posted By NGSfan
I am of the opinion that it is better to align to...

I am of the opinion that it is better to align to the genome. With STAR it can be done very quickly.

The question is, do you believe the transcriptome annotation is really complete? We know from...
Forum: Bioinformatics 02-19-2013, 04:30 AM
Replies: 218
Views: 118,350
Posted By NGSfan
That's good to know - I have been using 24...

That's good to know - I have been using 24 threads which is pretty fast, but I did try it on 8 threads and noticed it is not much faster at that level, especially if you include options for...
Forum: Bioinformatics 02-19-2013, 04:25 AM
Replies: 218
Views: 118,350
Posted By NGSfan
When the genome has a lot of contigs, I noticed...

When the genome has a lot of contigs, I noticed that STAR needs a lot more memory... how much do you have? I would recommend finding a machine with 256gb RAM if possible.
Forum: Bioinformatics 02-19-2013, 01:16 AM
Replies: 218
Views: 118,350
Posted By NGSfan
That is a great feature, Alex. I am looking...

That is a great feature, Alex. I am looking forward to it.

I currently convert cufflinks output into read counts for DESeq.

You read in the length and coverage columns from the ...
Forum: Bioinformatics 02-15-2013, 06:14 AM
Replies: 218
Views: 118,350
Posted By NGSfan
Yes, I am also happy with the results from STAR....

Yes, I am also happy with the results from STAR. It is incredibly fast and easy to use. I will test the shared memory option again now that I have my own workstation machine to play with.
...
Forum: Bioinformatics 02-06-2013, 02:20 AM
Replies: 5
Views: 3,089
Posted By NGSfan
They are different in what respect? Are...

They are different in what respect?

Are they reporting different locations of variants in the CDS transcript? Or are they different on the nucleotide/amino acid changes?

Or there are more...
Forum: RNA Sequencing 12-28-2012, 11:39 AM
Replies: 4
Views: 4,098
Posted By NGSfan
Alternatively, instead of pseudocounts to deal...

Alternatively, instead of pseudocounts to deal with some of the very small RPKMs generated by Cufflinks, you can also replace any RPKM < 0.0001 with 0 and this will give you the same values as...
Forum: RNA Sequencing 12-27-2012, 07:18 AM
Replies: 4
Views: 4,098
Posted By NGSfan
Solution found: For Cuffdiff/links 2.0.2 Make cufflinks FPKM match Cuffdiff FPKM

This is in reference to cuffdiff v2.0.2 (3522)

Hi, I have been banging my head on why cuffdiff and cufflinks have different FPKM calculations. I finally stumbled upon the settings to make...
Forum: Bioinformatics 11-16-2012, 01:40 PM
Replies: 12
Views: 5,079
Posted By NGSfan
You might want to look at our paper... it could...

You might want to look at our paper... it could be your sequencing depth is not sufficient enough to quantify your expression...

http://seqanswers.com/forums/showpost.php?p=44378&postcount=1
Forum: Bioinformatics 04-24-2012, 08:10 AM
Replies: 4
Views: 1,946
Posted By NGSfan
Very nice paper. Sometimes simpler is better.

Very nice paper.

Sometimes simpler is better.
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