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Forum: Sample Prep / Library Generation 05-17-2016, 09:49 AM
Replies: 4
Views: 1,657
Posted By jparsons
My main reason why not to use the spike-ins for...

My main reason why not to use the spike-ins for quantitative normalization is that I've never seen it demonstrated to work and that we are aware of biases that affect them - and these biases may vary...
Forum: Bioinformatics 05-13-2016, 01:50 PM
Replies: 4
Views: 1,325
Posted By jparsons
I've spent a good deal of time working on the...

I've spent a good deal of time working on the ERCCs and have a few points to make in their defense;

Yes, polyA selection biases affect some ERCC controls. There is no reason to believe this is...
Forum: Sample Prep / Library Generation 05-13-2016, 01:33 PM
Replies: 4
Views: 1,657
Posted By jparsons
A few points: RUV really doesn't work well...

A few points:

RUV really doesn't work well with ERCCs; it doesn't account for the fact that the controls have built-in differences. If you do want to use RUV for this type of purpose, you'll need...
Forum: Bioinformatics 06-29-2015, 01:30 PM
Replies: 15
Views: 7,863
Posted By jparsons
I'd be interested to hear more about your...

I'd be interested to hear more about your experience doing this. I have not heard many 'good' stories about normalizing to spike-ins in this space.
Forum: RNA Sequencing 05-14-2015, 03:40 PM
Replies: 1
Views: 848
Posted By jparsons
I would advocate for spike-in based normalization...

I would advocate for spike-in based normalization in this case. If you put spike-ins at 1:1, you can scale the remainder of reads accordingly. See Revisiting global gene expression analysis. Cell....
Forum: Bioinformatics 03-02-2015, 09:24 AM
Replies: 15
Views: 8,523
Posted By jparsons
In my opinion, ERCC spike-ins are uniquely...

In my opinion, ERCC spike-ins are uniquely situated to determine the mRNA:totalRNA ratio between samples, and are best used when you expect that the ratio is NOT equal. The Nature Biotech paper...
Forum: RNA Sequencing 12-04-2014, 10:07 AM
Replies: 3
Views: 1,460
Posted By jparsons
polyA selection is still filtering total RNA into...

polyA selection is still filtering total RNA into mRNA and must be accounted for.

It still sounds like you may have an ideal experiment for a mixture design.

Call your WT tissue A, your...
Forum: RNA Sequencing 12-02-2014, 12:01 PM
Replies: 3
Views: 1,460
Posted By jparsons
I have been working on a method to "unmix"...

I have been working on a method to "unmix" RNA-seq data from mixtures for awhile, and I think it may be of some help to your situation.

You would need to:
*Be able to collect a sample of your...
Forum: Bioinformatics 11-24-2014, 05:24 PM
Replies: 10
Views: 3,295
Posted By jparsons
I did a quick test of this today and saw no...

I did a quick test of this today and saw no obvious difference (except for a uniformly higher FPKM in "standard") between standard cufflinks and using --no-effective-length, as regards the reported...
Forum: Bioinformatics 11-18-2014, 11:07 AM
Replies: 1
Views: 740
Posted By jparsons
I have done some empirical mixture validation...

I have done some empirical mixture validation where I show that spike-ins do an excellent job of determining the different amounts of RNA per mixture component, assuming that you spike into a known...
Forum: Bioinformatics 11-10-2014, 03:34 PM
Replies: 3
Views: 1,362
Posted By jparsons
http://genomeinabottle.org/blog-entry/nist-na12878...

http://genomeinabottle.org/blog-entry/nist-na12878-highly-confident-integrated-genotype-calls-available-ftp-site
Forum: Illumina/Solexa 10-15-2014, 09:43 AM
Replies: 1
Views: 764
Posted By jparsons
The NIH has a lovely cost per genome graph, if...

The NIH has a lovely cost per genome graph, if you weren't already aware of it
http://www.genome.gov/sequencingcosts/
You can of course convert from per genome to per base if you so desire.
Forum: Illumina/Solexa 10-03-2014, 01:10 PM
Replies: 2
Views: 1,052
Posted By jparsons
State of the Art - RNAseq

This question really boils down to the following: If you were to do an RNA-seq experiment today (let's limit the sequencing to illumina) what protocols/kits would you use?

*For single-cell /...
Forum: Sample Prep / Library Generation 07-16-2014, 10:28 AM
Replies: 270
Views: 107,743
Posted By jparsons
From the discussions i've had with C1 users,...

From the discussions i've had with C1 users, Spike-ins for single cell are unfortunately very cell-type specific - you want to maintain a 3-5% amount of external RNA to cellular RNA, but the amount...
Forum: Bioinformatics 04-28-2014, 03:48 PM
Replies: 4
Views: 1,786
Posted By jparsons
Why are you using RPKM here? I'm not too...

Why are you using RPKM here?

I'm not too familiar with your study, but I'd think that you want to be looking at windows of very specific width (around the TSSs, +/- N nt) and therefore will be...
Forum: Bioinformatics 04-25-2014, 02:57 PM
Replies: 1
Views: 746
Posted By jparsons
Take a look at genomeinabottle.org Definitely...

Take a look at genomeinabottle.org
Definitely looking towards producing what you're asking for, and available in the case of NA12878:...
Forum: RNA Sequencing 04-21-2014, 11:22 AM
Replies: 1
Views: 2,592
Posted By jparsons
The practical difference between the mixes is...

The practical difference between the mixes is that most of the spike ins are at different concentrations between mixes.

You can use this difference to determine things - particularly detection...
Forum: Bioinformatics 04-21-2014, 11:05 AM
Replies: 2
Views: 1,366
Posted By jparsons
In my experience with very similar calculations,...

In my experience with very similar calculations, there is something about the RPKM normalization which makes ERCC spike-ins behave abnormally in cases with widely-disparate RNA content, which i...
Forum: Bioinformatics 03-20-2014, 03:33 PM
Replies: 15
Views: 5,529
Posted By jparsons
Awhile ago I did an analysis that compared the...

Awhile ago I did an analysis that compared the list of genes called as "Differentially expressed" across a set of mixture samples by a number of different algorithms. These mixture samples had some...
Forum: RNA Sequencing 01-21-2014, 10:40 AM
Replies: 5
Views: 2,012
Posted By jparsons
Illumina's human body map includes data for a...

Illumina's human body map includes data for a bunch of tissues (adipose adrenal brain breast colon heart kidney liver lung lymphnode ovary prostate muscle testes thyroid whiteblood) and has data out...
Forum: Bioinformatics 01-07-2014, 10:16 AM
Replies: 15
Views: 8,523
Posted By jparsons
The DESeq size factors assume that most things...

The DESeq size factors assume that most things will be 1:1, so the 1:1 sub pool would be a good fit.

That said, it's data analysis so there's nothing stopping you from doing it both ways - I'd be...
Forum: Bioinformatics 12-16-2013, 02:16 PM
Replies: 2
Views: 2,473
Posted By jparsons
Assa, You should be concerned with the...

Assa,

You should be concerned with the effect of the differential spike-in between the treatment and control samples. This effect may be quite minor or more serious, depending on the percentage...
Forum: Bioinformatics 10-22-2013, 09:35 AM
Replies: 10
Views: 2,973
Posted By jparsons
Is 'giving SAMstrt or something similar a try'...

Is 'giving SAMstrt or something similar a try' possible yet? As far as i can tell, the github is still just an unorganized pile of R scripts with next to 0 documentation.
Forum: General 09-19-2013, 12:33 PM
Replies: 8
Views: 3,128
Posted By jparsons
Until you're dealing with multiple runs worth of...

Until you're dealing with multiple runs worth of data, you're fine on a 'regular' machine, supposing it has a good amount (16+gb) of ram. There are only a few things that won't run on such a machine...
Forum: RNA Sequencing 08-29-2013, 01:02 PM
Replies: 11
Views: 10,197
Posted By jparsons
It didn't make sense to me either, but when I was...

It didn't make sense to me either, but when I was looking for places where there were discrepancies, that's what popped. If I had to hypothesize, i would think that the pseudo gene has unique...
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