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Forum: Sample Prep / Library Generation 12-12-2017, 09:55 AM
Replies: 10
Views: 1,602
Posted By jteeee2
That material will be fine for a total RNA Seq...

That material will be fine for a total RNA Seq approach. I wouldn't get concerned unless that value is above 10%. Also agree with pmiguel that the Qubit value can be misleading.
Forum: Illumina/Solexa 10-19-2017, 06:50 AM
Replies: 17
Views: 1,546
Posted By jteeee2
Glad to hear things are improving. Best of luck...

Glad to hear things are improving. Best of luck getting those last 23 samples made into libraries.
Forum: RNA Sequencing 09-21-2017, 05:58 AM
Replies: 15
Views: 2,794
Posted By jteeee2
Take a look at Kapa's RNA Seq documentation...

Take a look at Kapa's RNA Seq documentation (https://www.kapabiosystems.com/product-applications/products/next-generation-sequencing-2/rna-library-preparation-2/kapa-stranded-mrna-seq-kits/). They...
Forum: Illumina/Solexa 09-11-2017, 05:51 AM
Replies: 17
Views: 1,546
Posted By jteeee2
For FFPE samples, none of the adaptor-ligated...

For FFPE samples, none of the adaptor-ligated cDNA should be "reserved." All of it should move into the final PCR step. That's my opinion, and I'd be interested to hear what others thought. The P5...
Forum: Illumina/Solexa 09-06-2017, 12:46 PM
Replies: 17
Views: 1,546
Posted By jteeee2
I hope it works out for you. Regardless of...

I hope it works out for you. Regardless of outcome, I'd consider a using a different NGS provider :). Batch controls are incredibly important and often the only way to troubleshoot sample...
Forum: Illumina/Solexa 09-06-2017, 11:50 AM
Replies: 17
Views: 1,546
Posted By jteeee2
Also, did the core run any other samples...

Also, did the core run any other samples alongside yours? Did they include a batch control to verify the reagents were good? The fact that all of your samples performed so poorly makes me wonder if...
Forum: Illumina/Solexa 09-06-2017, 11:39 AM
Replies: 17
Views: 1,546
Posted By jteeee2
Glad to hear these were Qubit measurements. I...

Glad to hear these were Qubit measurements. I was initially concerned that they were Nanodrop readings and you were actually putting far less RNA into the library prep than you thought.

A quick...
Forum: Illumina/Solexa 09-06-2017, 11:26 AM
Replies: 17
Views: 1,546
Posted By jteeee2
That sounds like something Illumina would say. ...

That sounds like something Illumina would say. However, they do market these kits as being appropriate for FFPE material.

In the spreadsheets you attached, are those RNA concentrations nanodrop...
Forum: Illumina/Solexa 09-05-2017, 05:48 AM
Replies: 17
Views: 1,546
Posted By jteeee2
Generating RNA-Seq libraries from FFPE RNA can be...

Generating RNA-Seq libraries from FFPE RNA can be quite frustrating, so I definitely understand where you are coming from.

We haven't been very pleased with the RNA we get out of the Qiagen...
Forum: Sample Prep / Library Generation 08-24-2017, 11:44 AM
Replies: 1
Views: 949
Posted By jteeee2
You should be able to manually adjust the fast...

You should be able to manually adjust the fast region signal allowance under the advanced settings and this will clear the error, allowing for a RIN to be called.
Forum: Sample Prep / Library Generation 08-08-2017, 08:37 AM
Replies: 2
Views: 897
Posted By jteeee2
If you're really interested DNase treating, we...

If you're really interested DNase treating, we have had good success incorporating this product into the front end of several RNA Seq protocols (including SmartSeq). ...
Forum: Illumina/Solexa 06-08-2017, 01:37 PM
Replies: 3
Views: 907
Posted By jteeee2
For column cleanup of your already extracted RNA,...

For column cleanup of your already extracted RNA, I'd recommend the Zymo RNA Clean and Concentrator-5 workflow. Cheap, easy to use, and allows a smaller elution volume than compared to others (i.e....
Forum: Sample Prep / Library Generation 04-18-2017, 06:17 AM
Replies: 15
Views: 2,174
Posted By jteeee2
I agree with nucacidhunter about using the Pico...

I agree with nucacidhunter about using the Pico RNA assay on the Bioanalyzer. It would be interesting to see what this material looks like on the more sensitive assay.

Has this material been...
Forum: Sample Prep / Library Generation 04-17-2017, 02:26 PM
Replies: 15
Views: 2,174
Posted By jteeee2
Standard poly-A capture RNA-Seq protocols can go...

Standard poly-A capture RNA-Seq protocols can go down to about 100 ng of Total RNA starting material these days. Kapa (I don't work for Kapa, but I do like their NGS kits) sells a mRNA HyperPrep kit...
Forum: Illumina/Solexa 03-29-2017, 11:12 AM
Replies: 9
Views: 1,559
Posted By jteeee2
We haven't dealt with this specific situation...

We haven't dealt with this specific situation before, but with that length of identical sequence I would probably aim a little higher with the PhiX for the first attempt (20-30%). Would be...
Forum: Illumina/Solexa 03-29-2017, 07:33 AM
Replies: 9
Views: 1,559
Posted By jteeee2
What kind of libraries are they? Are you...

What kind of libraries are they? Are you expecting diversity to be low throughout the entire read length or just in specific parts?

We have had success with certain types of low diversity...
Forum: Sample Prep / Library Generation 01-24-2017, 06:10 AM
Replies: 6
Views: 2,082
Posted By jteeee2
I think you'll find it to be a pretty solid...

I think you'll find it to be a pretty solid workflow. You should be right around 90% exonic alignment rates with fresh frozen tissue. With FFPE material, we're in the 80-90% range. Best of luck!
Forum: Sample Prep / Library Generation 01-24-2017, 05:51 AM
Replies: 6
Views: 2,082
Posted By jteeee2
Unfortunately, I don't have any head to head...

Unfortunately, I don't have any head to head comparison data to share. The snoRNAs are actually targeted by probes within the RNA Access oligo pool, so in many cases there is nothing you can do do...
Forum: Sample Prep / Library Generation 11-14-2016, 06:14 AM
Replies: 6
Views: 2,082
Posted By jteeee2
We have seen several different snoRNA's soak up...

We have seen several different snoRNA's soak up reads in our analysis. We have been seeing the data improve recently by pre-heating the wash buffer during the second wash following captures. I...
Forum: Illumina/Solexa 09-30-2016, 07:28 AM
Replies: 16
Views: 4,318
Posted By jteeee2
We have both an Eppendorf 5075 TMX and a Perkin...

We have both an Eppendorf 5075 TMX and a Perkin Elmer Sciclone. In terms of NGS capability, the Sciclone is far superior to the Eppendorf 5075. As thermophile mentioned, the real headache is fine...
Forum: Sample Prep / Library Generation 08-19-2016, 12:17 PM
Replies: 6
Views: 2,082
Posted By jteeee2
TruSeq RNA Access Kit?

Has anyone been using the TruSeq RNA Access kit? We have been getting a snoRNA (SNORA63) soak up almost 10% of our reads from libraries we've generated with this kit. Seems to be present if we use...
Forum: Sample Prep / Library Generation 07-28-2016, 10:02 AM
Replies: 5
Views: 1,871
Posted By jteeee2
Do you have any control RNA material that you've...

Do you have any control RNA material that you've made into a library using this workflow? The only way to avoid a situation like this in the future is to always include some sort of batch control. ...
Forum: Sample Prep / Library Generation 07-28-2016, 05:22 AM
Replies: 5
Views: 1,871
Posted By jteeee2
There are a lot of possible reasons your yields...

There are a lot of possible reasons your yields may be lower than what you have seen previously. In a perfect world, we would see consistent recoveries from one RNA-Seq experiment to the next, but...
Forum: Sample Prep / Library Generation 07-27-2016, 09:22 AM
Replies: 5
Views: 1,871
Posted By jteeee2
What type of RNA-Seq are you doing? Poly-A...

What type of RNA-Seq are you doing? Poly-A enriched? rRNA depleted?

How much Total RNA did you start with? Was it of high quality (RIN score, etc.)?
Forum: Sample Prep / Library Generation 06-10-2016, 06:57 AM
Replies: 3
Views: 1,109
Posted By jteeee2
Thanks for attaching these. The only pertinent...

Thanks for attaching these. The only pertinent comment I have about the traces is that the average library size is a bit larger than we commonly see for captures. Was genomic DNA used as the...
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