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Forum: Sample Prep / Library Generation 07-23-2014, 09:10 AM
Replies: 4
Views: 1,566
Posted By ZWB
I do mixed population (host/pathogen) RNA-Seq...

I do mixed population (host/pathogen) RNA-Seq often and we always use the Direct-Zol kit from Zymo to do our extractions. This kit combines the advantages of Trizol with the ease of a column and...
Forum: Genomic Resequencing 03-19-2014, 07:47 AM
Replies: 3
Views: 3,641
Posted By ZWB
ECO, Thanks for the great idea! This will...

ECO,

Thanks for the great idea! This will save me a lot of headache as I prepare for some higher throughput experiments I'm planning.

Zach
Forum: Sample Prep / Library Generation 03-19-2014, 07:42 AM
Replies: 4
Views: 3,289
Posted By ZWB
We find that the primary cause of concatamers...

We find that the primary cause of concatamers (assuming the RNA is abundant and of high quality) is over amplification in the final PCR step. I would try decreasing the number of PCR cycles....
Forum: Metagenomics 03-14-2014, 10:00 AM
Replies: 2
Views: 2,003
Posted By ZWB
I think in most cases you will have a hard time...

I think in most cases you will have a hard time getting to the species level using just the V4 region. With Illumina's 600 cycle kit for the MiSeq it's now possible to sequence V1-V3 or other longer...
Forum: Sample Prep / Library Generation 01-15-2014, 08:12 AM
Replies: 2
Views: 1,663
Posted By ZWB
I've never run the SOLID protocol, but for our...

I've never run the SOLID protocol, but for our Illumina protocols we use the NEB Next RNA fragmentation kit. It's a chemical rather than an enzymatic fragmentation. We end up with average sizes...
Forum: Sample Prep / Library Generation 11-25-2013, 07:55 AM
Replies: 18
Views: 11,038
Posted By ZWB
In my experience, the double hump is the result...

In my experience, the double hump is the result of over amplification during your final PCR step. I would try 12 cycles instead of 14 and see if that gets rid of the second hump.
Forum: Sample Prep / Library Generation 09-27-2013, 07:48 AM
Replies: 1
Views: 2,084
Posted By ZWB
You can deplete rRNA at the cDNA level if you use...

You can deplete rRNA at the cDNA level if you use a technique like DSN (double-stranded nuclease) or HAC (hydroxyapatite chromatography). We published a paper a while back about HAC, showing high...
Forum: Illumina/Solexa 08-02-2013, 11:04 AM
Replies: 4
Views: 5,130
Posted By ZWB
We pretty much use the exact same protocol as...

We pretty much use the exact same protocol as you. We found that the best enzyme mix to use is FailSafe with Buffer E (EpiCentre). I've heard rumors that the buffer in the Nextera PCR mix is in fact...
Forum: Sample Prep / Library Generation 07-08-2013, 01:50 PM
Replies: 14
Views: 11,800
Posted By ZWB
I've had similar problems in the past with failed...

I've had similar problems in the past with failed tagmentation and it was always due to contaminants/inhibitors in the sample. In my experience, the MoBio power clean kit seems to do the best job...
Forum: Sample Prep / Library Generation 06-20-2013, 07:43 AM
Replies: 3
Views: 3,803
Posted By ZWB
I've been doing 16S amplicon sequencing lately...

I've been doing 16S amplicon sequencing lately and I've found that quantifying by Qubit and pooling works fine, although not quite as good as qPCR. On most runs I see only a slight (+/- 2%)...
Forum: Illumina/Solexa 06-19-2013, 07:33 AM
Replies: 2
Views: 1,788
Posted By ZWB
While 6 cycles does seem really low, I've done as...

While 6 cycles does seem really low, I've done as few as 5 cycles with good results. If you are worried you could do an extra cycle or two (no more that 8 total) and the library should still be OK....
Forum: Illumina/Solexa 06-03-2013, 03:56 PM
Replies: 7
Views: 1,674
Posted By ZWB
It's harder not to isolate 16S rRNA from a...

It's harder not to isolate 16S rRNA from a sample. >90% of the bacterial transcripts will be rRNA. You probably don't need to do anything special to get rRNA, although you could make 16S probes to...
Forum: Illumina/Solexa 06-03-2013, 09:27 AM
Replies: 7
Views: 1,674
Posted By ZWB
Jamie, I've tried that before and it works...

Jamie,

I've tried that before and it works fairly well. We called it negative capture for the reason you described (capturing what you don't want). The problem is getting the most similar host RNA...
Forum: Illumina/Solexa 06-03-2013, 08:15 AM
Replies: 7
Views: 1,674
Posted By ZWB
We've developed a technique that separates host...

We've developed a technique that separates host transcripts from bacterial transcripts, but right now it only works if the organism of interest is culturable. You may be able to make probes using...
Forum: Sample Prep / Library Generation 05-16-2013, 07:46 AM
Replies: 35
Views: 20,459
Posted By ZWB
This is a recently published technique that we...

This is a recently published technique that we developed to make directional RNA-Seq libraries. It's similar to one of the techniques in the Levin et al paper, but we made some significant...
Forum: Illumina/Solexa 05-07-2013, 10:14 AM
Replies: 12
Views: 28,260
Posted By ZWB
This is a recently published, directional RNA...

This is a recently published, directional RNA library prep protocol that goes down to as low as 10ng of total RNA (or rRNA depleted RNA) using random priming. We have also tried 5ng of rRNA depleted...
Forum: RNA Sequencing 04-29-2013, 07:56 AM
Replies: 2
Views: 1,468
Posted By ZWB
Hello B, I've been doing some bacterial...

Hello B,

I've been doing some bacterial transcriptome analysis from human clinical samples and found that the portion of bacterial RNA is very small. Typically we find ~0.01% of the reads map to...
Forum: RNA Sequencing 03-15-2013, 08:01 AM
Replies: 3
Views: 1,445
Posted By ZWB
I'm working on a similar problem. We were able to...

I'm working on a similar problem. We were able to find >200 DE genes in our system and obviously we couldn't do qRT-PCR on all of them. We chose to look at 10 genes (5 up-regulated and 5...
Forum: Sample Prep / Library Generation 03-05-2013, 03:46 PM
Replies: 5
Views: 4,618
Posted By ZWB
Have you tried using less enzyme? I think using...

Have you tried using less enzyme? I think using less transposon would be a more effective way to increase fragment size than decreasing (the already short) reaction time.
Forum: Sample Prep / Library Generation 03-04-2013, 07:38 AM
Replies: 10
Views: 9,334
Posted By ZWB
Hi Chris, We've had similar problems with...

Hi Chris,

We've had similar problems with determining concentrations. The problem may actually be the nanodrop rather than your protocol. We switched to a Qubit and get much more reliable results....
Forum: Sample Prep / Library Generation 06-28-2012, 09:57 AM
Replies: 5
Views: 4,480
Posted By ZWB
We find that the most highly expressed genes are...

We find that the most highly expressed genes are still the most highly expressed genes after DSN treamtent. So if you look at a metric like the top 10 most highly expressed genes before and after DSN...
Forum: Sample Prep / Library Generation 06-27-2012, 09:22 AM
Replies: 5
Views: 4,480
Posted By ZWB
Hello, We have had very good luck removing...

Hello,

We have had very good luck removing rRNA from degraded samples using DSN treatment. In degraded samples we find that DSN works much better than Ribo-Zero often dropping the final rRNA to...
Forum: Sample Prep / Library Generation 06-18-2012, 08:33 AM
Replies: 1
Views: 1,364
Posted By ZWB
I've been successful using the NEBNext...

I've been successful using the NEBNext fragmentation kit with an incubation of 3 minutes instead of 5. Make sure to use only 200ng/rnx (or less) and pool multiple rxns during cleanup. This has worked...
Forum: Illumina/Solexa 06-07-2012, 08:04 AM
Replies: 10
Views: 10,236
Posted By ZWB
We've also had this issue on multiple runs. Try...

We've also had this issue on multiple runs. Try running the volume test. When this happened to us it was typically a result of a valve failure.
Forum: Sample Prep / Library Generation 06-04-2012, 09:23 AM
Replies: 4
Views: 2,329
Posted By ZWB
Try using NEBNext RNA fragmentation buffer. We do...

Try using NEBNext RNA fragmentation buffer. We do a 3 min incubation and get a pretty good size distribution for 100 PE runs
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