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Forum: Pacific Biosciences 06-22-2017, 11:17 AM
Replies: 9
Views: 2,092
Posted By docbio
I do have access to a MinION, so I am familiar...

I do have access to a MinION, so I am familiar with it. Things are much much better with the R9.4 flow cells and we're still getting our protocols optimized for making the 1D libraries, interested in...
Forum: Pacific Biosciences 06-20-2017, 07:44 AM
Replies: 9
Views: 2,092
Posted By docbio
Will do, although not sure how forthcoming...

Will do, although not sure how forthcoming they'll be on their costs vs what they charge... some sequencing centers treat that as a bit of proprietary information.

Just to be clear, I'm not...
Forum: Pacific Biosciences 06-20-2017, 06:15 AM
Replies: 9
Views: 2,092
Posted By docbio
Question Sequel Sequencing Costs - Microbial Genomes

Howdy,

Tried to post this once, and it disappeared.

I have been tasked with assembling a per genome cost of Sequel sequencing (assuming we own the instrument already). We are primarily...
Forum: Illumina/Solexa 01-22-2015, 12:54 PM
Replies: 5
Views: 1,588
Posted By docbio
FYI, just as a follow up, we found that half...

FYI, just as a follow up, we found that half reactions and even one quarter reactions work very well with NexteraXT in our hands, although the one quarter reaction size requires very precise...
Forum: RNA Sequencing 01-22-2015, 12:48 PM
Replies: 0
Views: 620
Posted By docbio
Smile RNAseq - Alternatives to SeqScript?

Howdy all,

Looking for lower cost alternatives to SeqScript. I realize a big portion of the cost is tied up in the Ribo Zero depletion reagent, so I'm also considering lower cost alternatives to...
Forum: Illumina/Solexa 02-27-2014, 07:33 AM
Replies: 5
Views: 1,588
Posted By docbio
Interesting. This is useful info, I'm assuming...

Interesting. This is useful info, I'm assuming the Fluidigm C1 protocol is online? I'm sure the concentration of DNA is critical to the tagmentation reaction. If we could move to half reactions it...
Forum: Sample Prep / Library Generation 02-27-2014, 07:27 AM
Replies: 16
Views: 29,597
Posted By docbio
Have you heard an estimated price? Based upon the...

Have you heard an estimated price? Based upon the difference between 24 and 96, I'm expecting it to be a fairly linear increase...
Forum: Illumina/Solexa 02-26-2014, 04:37 PM
Replies: 5
Views: 1,588
Posted By docbio
Half reactions for NexteraXT

Howdy all,

Wondering if anyone has had any success running "half reactions" of NexteraXT libraries or otherwise stretching those reagents? At the end of the library cleanup, if memory serves you...
Forum: Sample Prep / Library Generation 02-26-2014, 04:32 PM
Replies: 16
Views: 29,597
Posted By docbio
So I hear from my Illumina rep that 384 index...

So I hear from my Illumina rep that 384 index combinations for NexteraXT should be coming in April 2014. We will see...
Forum: Core Facilities 02-26-2014, 04:30 PM
Replies: 18
Views: 12,480
Posted By docbio
Can't help you with the first part of the...

Can't help you with the first part of the question, but in terms of bead carryover I've found it can often be addressed by slowing down the speed of the pipetting either in automated or manual...
Forum: Illumina/Solexa 05-10-2013, 12:04 PM
Replies: 7
Views: 2,248
Posted By docbio
Also want to point out we designed our protocol...

Also want to point out we designed our protocol with two interventions to do the tagmentation step and the amplification step off robot in a thermocycler. We didn't trust the TMX module to hold the...
Forum: Sample Prep / Library Generation 05-10-2013, 11:28 AM
Replies: 16
Views: 29,597
Posted By docbio
Ahhh, but that's the rub. It's not just a...

Ahhh, but that's the rub. It's not just a "standard oligo primer" - the adapters have a forked structure and there is some proprietary modification to the adapters that Illumina's not talking about....
Forum: Sample Prep / Library Generation 05-09-2013, 08:22 PM
Replies: 16
Views: 29,597
Posted By docbio
Multiplexing >96 Samples with NexteraXT

Ok, so the current NexteraXT dual-indexing adapters allow for 96 combinations using a 8x12 setup. We routinely run 96 samples per lane, and honestly, we'd like to add more.

The dual-index system...
Forum: Illumina/Solexa 05-09-2013, 08:12 PM
Replies: 7
Views: 2,248
Posted By docbio
We have an Eppendorf Epmotion 5075 TMX in house...

We have an Eppendorf Epmotion 5075 TMX in house now. We've been working with their technical reps to develop the NexteraXT protocol for the platform and optimize it. It has required a little bit of...
Forum: Illumina/Solexa 02-14-2013, 08:24 PM
Replies: 18
Views: 12,728
Posted By docbio
Matt, Sorry I can't be of much help. I'm...

Matt,

Sorry I can't be of much help. I'm sending my samples to a core facility and they flat out refuse to mix chemistries on a flow cell. I had been led to believe that it wasn't even possible to...
Forum: Illumina/Solexa 11-16-2012, 12:58 PM
Replies: 7
Views: 2,248
Posted By docbio
Automation for NexteraXT Library Prep

Curious if anyone has any experience automation platforms and the NexteraXT library preparation kits.

We're looking at several possibilities, including an epMotion 5075 TMX. I've also recently...
Forum: Illumina/Solexa 11-16-2012, 12:50 PM
Replies: 18
Views: 12,728
Posted By docbio
Sorry I didn't see this post earlier. We've been...

Sorry I didn't see this post earlier. We've been running NexteraXT libraries on the HiSeq with good results. Following the recommendations of Illumina, we stop after library cleanup (no bead based...
Forum: Core Facilities 11-16-2012, 12:34 PM
Replies: 18
Views: 12,480
Posted By docbio
I know this thread is old, but curious if anyone...

I know this thread is old, but curious if anyone has any updates on their experience with the 5075, in particular the 5075 TMX platform, or any competitor automation platforms (SciClone, etc) for NGS...
Forum: Sample Prep / Library Generation 08-24-2012, 10:11 AM
Replies: 4
Views: 3,183
Posted By docbio
My pool volumes wound up being rather large...

My pool volumes wound up being rather large (~500uL) and I didn't have any un-expired AmpureXP on hand, so I decided to call an audible and try an Amicon 0.5ml Ultracel-30K concentrator that we had...
Forum: Sample Prep / Library Generation 08-23-2012, 10:30 AM
Replies: 4
Views: 3,183
Posted By docbio
I have AmpureXP beads in house but they are a...

I have AmpureXP beads in house but they are a couple of months out of date - not sure how quickly they degrade but not sure I want to risk it. I have fresh AmpureXP on order.

If I were to go the...
Forum: Sample Prep / Library Generation 08-22-2012, 01:16 PM
Replies: 4
Views: 3,183
Posted By docbio
Concentrating NexteraXT libraries

Just wondering if anyone has any experience with this. I am still fairly new to preparing NexteraXT libraries. I have had good luck with the bead based normalization and running pools on the MiSeq,...
Forum: Sample Prep / Library Generation 08-22-2012, 01:08 PM
Replies: 5
Views: 6,466
Posted By docbio
What about the expiration date? I am generally...

What about the expiration date? I am generally curious if people have any experience with "expired" Ampure XP beads? I have been inadvertently using some which are just a couple of months out of date...
Forum: Illumina/Solexa 07-19-2012, 10:57 AM
Replies: 18
Views: 6,169
Posted By docbio
I can't tell you the concentration you should...

I can't tell you the concentration you should expect but I feel like the libraries coming out of the Nextera are fairly dilute, so your concentration may not be off.

When I talked to tech support...
Forum: Illumina/Solexa 07-19-2012, 08:25 AM
Replies: 10
Views: 9,024
Posted By docbio
Cluster Generation Failure

Several things:

1) We also had a valve failure, this is supposedly due to crystals forming in one of the valves and grinding it down - this led to the new wash protocol recommendations in MCS...
Forum: Illumina/Solexa 07-19-2012, 08:06 AM
Replies: 18
Views: 6,169
Posted By docbio
Adapter trimming?

Incidentally, are the N's all appearing at the end of the reads? That is, do you have strings of NNNNNNN? If so, did you leave "Trim Adapter Sequences?" turned on when you made your sample sheet in...
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