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Forum: Illumina/Solexa 08-04-2015, 12:38 AM
Replies: 9
Views: 9,297
Posted By Asaf
I emailed Illumina's representatives here in...

I emailed Illumina's representatives here in Israel but didn't get an answer. I think that the explanation I gave above is reasonable (maybe low efficiency of RT in the cluster?). With v.2 chemistry...
Forum: Illumina/Solexa 02-23-2015, 02:52 AM
Replies: 2
Views: 3,103
Posted By Asaf
I didn't try to contact them yet, I'm trying to...

I didn't try to contact them yet, I'm trying to see if it's common or related to the library construction first. The libraries have a 9 bp barcode and they are bacterial rRNA depleted transcriptomes...
Forum: Illumina/Solexa 02-22-2015, 07:32 AM
Replies: 2
Views: 3,103
Posted By Asaf
Unhappy Nextseq500 N's from position 25

Hi,
I've been seeing a lot of Nextseq500 runs lately and I found a very disturbing phenomenon. In a lot of the runs I start seeing a lot of N's from cycle 25 onward. The FastQC looks like this:...
Forum: Sample Prep / Library Generation 12-25-2014, 01:02 AM
Replies: 68
Views: 26,142
Posted By Asaf
Some insights from our test nextseq run. We...

Some insights from our test nextseq run.
We took some RNA (8 RNAseq libraries), prepared the libraries after RiboZero, fragmentation and size selection. The process was indeed shorter (1.5 days) and...
Forum: Bioinformatics 12-21-2014, 05:52 AM
Replies: 4
Views: 1,871
Posted By Asaf
Thanks again, We have 2 replicates of all 4...

Thanks again,
We have 2 replicates of all 4 libraries so we should have enough, I do get significant genes (13) and some of them even makes sense biologically :).
Forum: Bioinformatics 12-18-2014, 05:39 AM
Replies: 4
Views: 1,871
Posted By Asaf
Thanks Mike, it's much clearer now. However,...

Thanks Mike, it's much clearer now.
However, after doing some thinking since I wrote the first post I now think that the formula should be:
full=~group+condition+group:condition,...
Forum: Bioinformatics 12-18-2014, 03:08 AM
Replies: 4
Views: 1,871
Posted By Asaf
DESeq2 LRT insights

Hi,
We're working on a RNA binding protein and want to figure out if it binds different RNAs in 2 different conditions. We did an immunoprecipitation (IP) for the protein and prepared the RNA that...
Forum: Sample Prep / Library Generation 12-14-2014, 12:34 AM
Replies: 4
Views: 2,797
Posted By Asaf
If you're still skeptic you can always take some...

If you're still skeptic you can always take some of the RNA and PCR your mRNA of interest to make sure it's there. However, you shouldn't worry as MU Core said, there are no peaks for mRNAs in...
Forum: Illumina/Solexa 12-10-2014, 10:18 PM
Replies: 3
Views: 2,083
Posted By Asaf
Alternatively, you can use UMI - unique molecule...

Alternatively, you can use UMI - unique molecule identifiers. Each adapter that you ligate contains abarcode (of the library) and a short sequence (4-6 nt) unique for that molecule. After...
Forum: General 12-10-2014, 03:51 AM
Replies: 16
Views: 4,296
Posted By Asaf
I opened Lehninger (Principles of biochemistry,...

I opened Lehninger (Principles of biochemistry, 4th ed.) on page 293. I quote:

C is deaminated to U in a rate of 10^-7 in 24 hours, which means 100 mutations a day for a mammalian cell which would...
Forum: Introductions 12-09-2014, 05:34 AM
Replies: 8
Views: 2,263
Posted By Asaf
We got an offer from SQREAM...

We got an offer from SQREAM (http://www.sqreamtech.com/), they develop a fast GPU based SQL server with the storage of infinidat (http://www.infinidat.com/).
They have a niche for biological data...
Forum: RNA Sequencing 12-09-2014, 05:26 AM
Replies: 1
Views: 2,364
Posted By Asaf
Normalization is needed when you want to compare...

Normalization is needed when you want to compare two libraries of RNAseq. Essentially you would want to know if the number of reads mapped to a gene in two libraries is different (or significantly...
Forum: Bioinformatics 11-19-2014, 11:37 PM
Replies: 2
Views: 1,113
Posted By Asaf
Look at iTOL help page under "Uploading and...

Look at iTOL help page under "Uploading and working with your own trees" to display bars. I think the only way to mark a group is by coloring it. After you upload the tree click on "Define color...
Forum: Sample Prep / Library Generation 11-18-2014, 01:15 AM
Replies: 68
Views: 26,142
Posted By Asaf
Thanks! We'll use this protocol for our next RNA...

Thanks! We'll use this protocol for our next RNA library construction.
Forum: Sample Prep / Library Generation 11-16-2014, 01:53 PM
Replies: 68
Views: 26,142
Posted By Asaf
This is a very nice protocol, thanks for sharing....

This is a very nice protocol, thanks for sharing.
I have two questions about the 5' ends of RNA libraries:
1. Why is there a bias in the 5' towards G (and not A)
2. Are there always 3 G's at the...
Forum: RNA Sequencing 11-01-2014, 12:51 PM
Replies: 5
Views: 1,705
Posted By Asaf
Thanks, it's good to hear we're not alone. A lot...

Thanks, it's good to hear we're not alone. A lot of steps were different between the two protocols including cDNA synthesis which explains the differences. I'll just add another factor that we...
Forum: RNA Sequencing 10-30-2014, 12:16 AM
Replies: 5
Views: 1,705
Posted By Asaf
Correlation between library construction protocols

Hi,
We've done some experiments in out lab and from the same RNA constructed libraries using two different protocols. To our surprise the correlation between the two protocols was lower than...
Forum: RNA Sequencing 10-07-2014, 03:20 AM
Replies: 3
Views: 1,368
Posted By Asaf
Do you see a correlation between the p-values of...

Do you see a correlation between the p-values of all the genes between the two pipelines? Do the differentially expressed genes in the two pipelines overlap?
Forum: RNA Sequencing 10-06-2014, 01:32 AM
Replies: 6
Views: 2,177
Posted By Asaf
Maybe you should compare pairs

You're going to stumble into many normalization issues here. I'm having trouble comparing experiments from the same lab in two different experimental settings, let alone different labs and bacteria....
Forum: Illumina/Solexa 07-20-2014, 11:28 AM
Replies: 9
Views: 9,297
Posted By Asaf
poly-G in NextSeq

Hi,
I just received NextSeq paired-end results (45 bp 1st read and 40 bp second read) and I noticed (using fastQC) that about 1-2% of the second read is poly-G. I known that G has no "color" so it...
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