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Forum: RNA Sequencing 04-09-2019, 01:19 PM
Replies: 0
Views: 1,895
Posted By JeremyDay
No bam from kallisto or Salmon after piping into samtools

Hi all,

I am trying to generate the pseudo bam files from both Kallisto v45 and Salmon v13 and can't get either to pipe into Samtools and generate a non-emtpy file. I think I am more currently...
Forum: RNA Sequencing 10-09-2018, 12:03 PM
Replies: 2
Views: 2,234
Posted By JeremyDay
Unfortunately, I never found a good solution. I...

Unfortunately, I never found a good solution. I developed a method on our FXP for the Zymo kit. It worked very well for cultured cells, but the second we put clinical samples on, we found that it was...
Forum: Epigenetics 05-18-2018, 12:24 PM
Replies: 17
Views: 12,595
Posted By JeremyDay
I hope you mean 100ng of DNA

I hope you mean 100ng of DNA
Forum: RNA Sequencing 01-15-2015, 01:57 PM
Replies: 2
Views: 2,234
Posted By JeremyDay
High throughput RNA extractions - best methods/kits?

Hi All,

We have a large number of RNA samples coming (most likely in Trizol) this year. I'd like to streamline and get some consistency by deciding on a product that gives good quality (260/280,...
Forum: Sample Prep / Library Generation 07-11-2014, 04:07 PM
Replies: 15
Views: 13,558
Posted By JeremyDay
Except that he's posted this somewhere else and...

Except that he's posted this somewhere else and forgot to mention here that there are barcodes ligated to the ends of these samples.
Forum: Sample Prep / Library Generation 07-11-2014, 01:19 PM
Replies: 15
Views: 13,558
Posted By JeremyDay
Not possible with Nextera. You could either add...

Not possible with Nextera. You could either add the adapter sequences with PCR or ligate adapters to the ends of your cDNA using a standard Truseq Nano, a kit from NEB, or another supplier. In the...
Forum: Illumina/Solexa 06-27-2014, 12:35 AM
Replies: 18
Views: 8,426
Posted By JeremyDay
I agree that matching your adapter to input...

I agree that matching your adapter to input molarity should solve any problems with adapter dimers if you ligate. However, out of curiosity, have you tried doing the same with your primers? Primers...
Forum: General 06-24-2014, 04:00 PM
Replies: 10
Views: 5,876
Posted By JeremyDay
50-100 samples of miRNA and RRBS, will cost big...

50-100 samples of miRNA and RRBS, will cost big bucks if you try to do it on a Miseq! Library prep is a little more complicated to find costs, but... there is an update coming soon for Rapid Runs on...
Forum: Epigenetics 06-24-2014, 03:42 PM
Replies: 17
Views: 12,595
Posted By JeremyDay
Hi rosatoc, We struggled with RRBS for a...

Hi rosatoc,

We struggled with RRBS for a couple of months. The input was DNA from 64-cell embryos, so ~200pg. Eventually we ended up using Nugen's ultra-low Ovation kit. We never saw the...
Forum: Illumina/Solexa 04-21-2014, 03:00 PM
Replies: 15
Views: 5,510
Posted By JeremyDay
Is there is a chance it could be sample related....

Is there is a chance it could be sample related. Assuming these are either HT or Nextera based, are they different preps (another company? someone else doing the preps?) than say your single indexed...
Forum: Ion Torrent 04-02-2014, 04:33 PM
Replies: 4
Views: 3,556
Posted By JeremyDay
Just to add to above (or amend) I think 10-15...

Just to add to above (or amend) I think 10-15 cycles is prob over kill. If you have an established library already, even 6 cycles will yield 97% new ends. You can start with 5-10ng and 6 cycles will...
Forum: Illumina/Solexa 03-14-2014, 01:33 PM
Replies: 2
Views: 1,242
Posted By JeremyDay
Oh, and dont forget to keep the T there. Dont...

Oh, and dont forget to keep the T there. Dont replace it with NN. If using standard illumina seq primers, it needs to be there.
Forum: Illumina/Solexa 03-13-2014, 04:16 PM
Replies: 2
Views: 1,242
Posted By JeremyDay
instead of a T overhang, adapters with degenerate...

instead of a T overhang, adapters with degenerate di-nuleotide overhangs? In which case you can just add NN onto your primers when you order them
Forum: Illumina/Solexa 03-13-2014, 03:37 PM
Replies: 55
Views: 32,783
Posted By JeremyDay
FYI. The latest RTA software is much better than...

FYI. The latest RTA software is much better than a year ago. I am currently running 3 amplicons with 10% phiX at 700k/mm^2. It's a nano, but Im getting 1M reads with 95% passing filter and 99% Q30. I...
Forum: Sample Prep / Library Generation 01-16-2014, 12:56 PM
Replies: 28
Views: 12,112
Posted By JeremyDay
I know this thread is a bit older now, but I am...

I know this thread is a bit older now, but I am interested in other alternatives as well. Kapa is darn expensive, but IS consistent. I think I'll need to play with different concentrations of...
Forum: Sample Prep / Library Generation 11-08-2013, 04:11 PM
Replies: 2
Views: 1,913
Posted By JeremyDay
First have you tried eluting off the columns with...

First have you tried eluting off the columns with smaller volumes (25x2)? And use prewarmed Elution buffer.

Depending on your lab, it may be a large investment, but quant-it picogreen is pretty...
Forum: Sample Prep / Library Generation 11-08-2013, 02:15 PM
Replies: 2
Views: 2,421
Posted By JeremyDay
The final normalization is for pooling...

The final normalization is for pooling multiplexed samples. Its main purpose is to pool each individual library at equimolar concentrations. Illumina normally advises normalizing the libraries to the...
Forum: Sample Prep / Library Generation 11-08-2013, 01:50 PM
Replies: 3
Views: 4,087
Posted By JeremyDay
Also, what is the size of DNA for starting...

Also, what is the size of DNA for starting material? Genomic? Fragmented?
Forum: Sample Prep / Library Generation 11-08-2013, 01:49 PM
Replies: 3
Views: 4,087
Posted By JeremyDay
Can you provide volumes of each reagent/sample? ...

Can you provide volumes of each reagent/sample?
Vol Starting material=
Vol Ampure added 1st sel=
Volume of Ampure the supernatant is added to=

Starting material is only DNA and H2O or TE,...
Forum: Sample Prep / Library Generation 11-08-2013, 01:37 PM
Replies: 1
Views: 1,322
Posted By JeremyDay
Hi Noa- My guess is genomic DNA...

Hi Noa-

My guess is genomic DNA contamination. Did you DNase treat it when on the column?
Forum: Bioinformatics 01-07-2013, 02:55 PM
Replies: 13
Views: 8,293
Posted By JeremyDay
Thanks kbhit! I'll have to take a look at this. ...

Thanks kbhit! I'll have to take a look at this.

Do you mind if I ask what you are getting for mappability using Shrimp2?

I appreciate your input. Finding a proper pipeline for Solid data is...
Forum: Bioinformatics 01-07-2013, 12:04 PM
Replies: 13
Views: 8,293
Posted By JeremyDay
Solid mapping

KBhit- Do you mind elaborating on this? I have searched and searched for a better way to map Solid data. When we use Lifescope compared to something like Bowtie, its a difference of 90% and 60%. No...
Forum: Sample Prep / Library Generation 06-15-2012, 12:54 PM
Replies: 16
Views: 9,935
Posted By JeremyDay
qPCR

We use Solid Taqman assays for our Solid machines. They have such kits for Illumina also. If I remember correctly, you can buy them from Kappa and Agilent. They are expensive, but then again so isn't...
Forum: RNA Sequencing 06-15-2012, 12:47 PM
Replies: 5
Views: 4,414
Posted By JeremyDay
Tophat 1

I am not a Tophat user, but I have heard from others that Tophat 2.0 changed from Tophat 1 in the sense that it maps only to annotated references, which reduces mapability. Maybe try Tophat 1? Some...
Forum: SOLiD 04-11-2012, 04:37 PM
Replies: 4
Views: 3,398
Posted By JeremyDay
light source

Hi RickC7- I was wondering if you ever found an alternative source for the light, or if we must go through Lifetech? Thanks!
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