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Forum: Sample Prep / Library Generation 02-29-2016, 05:22 PM
Replies: 1
Views: 1,480
Posted By Ingeneious
AMPure XP on Hamilton STARlet

Does anyone have a method they would be willing to share? Hamilton's shared resources are limited. I have something that I am working on adapting, but it will take some time.

Semi-related...
Forum: Sample Prep / Library Generation 01-30-2016, 05:18 PM
Replies: 17
Views: 15,475
Posted By Ingeneious
I think that is it, other than the walk away...

I think that is it, other than the walk away capability. Pricing is about $5k more than the old system. Uses the same tapes and allows selecting specific wells. Not sure if that requires two at a...
Forum: Illumina/Solexa 01-26-2016, 10:34 AM
Replies: 31
Views: 11,684
Posted By Ingeneious
There is a cost / read advantage if you are doing...

There is a cost / read advantage if you are doing 2 x 150 PE sequencing. 50% of the capital cost of the MiSeq and the NextSeq-esque wash system also means less maintenance than the MiSeq.

Overally...
Forum: Illumina/Solexa 12-17-2015, 10:21 AM
Replies: 5
Views: 1,250
Posted By Ingeneious
Thanks for the advice. Have you sequenced with...

Thanks for the advice. Have you sequenced with that much? I have seen that recommendation on the boards, but wondering if I could get away with more.

This would be for more than a single...
Forum: Illumina/Solexa 12-16-2015, 11:31 AM
Replies: 5
Views: 1,250
Posted By Ingeneious
Maximum concentration of adapter dimers?

I have around 10% adapter dimers, relative to the molarity of my target product, in my final library. I am trying to avoid another cleanup to reduce costs. What are the maximum amount of adapter...
Forum: Illumina/Solexa 12-02-2015, 12:57 AM
Replies: 13
Views: 5,316
Posted By Ingeneious
Thanks for all the informative replies. Dark...

Thanks for all the informative replies. Dark cycles - will try pushing the limit, if I ever need to in the future. I have gone over the 25 extra cycles before and did not have noticeable issues....
Forum: Illumina/Solexa 11-26-2015, 07:14 AM
Replies: 13
Views: 5,316
Posted By Ingeneious
Dark cycles

According to the MiSeq system user guide, the kits contain an additional 26 cycles of reagents (i.e. 50 cycle kit contains 76 cycles of reagents, 300 cycle kit contains 326 cycles of reagents, etc.)....
Forum: Sample Prep / Library Generation 11-26-2015, 06:58 AM
Replies: 6
Views: 2,300
Posted By Ingeneious
Thanks for your input, nucacidhunter.

Thanks for your input, nucacidhunter.
Forum: Sample Prep / Library Generation 11-24-2015, 07:36 AM
Replies: 6
Views: 2,300
Posted By Ingeneious
This is ligation-based amplicon sequencing, so no...

This is ligation-based amplicon sequencing, so no shotgun involved.

What could cause read2 dropout? %Q30 scores were all >90%.
Forum: Sample Prep / Library Generation 11-23-2015, 11:30 PM
Replies: 6
Views: 2,300
Posted By Ingeneious
TruSeq DNA HT, 2 x 8bp, 200bp, 2 x 150 PE.

TruSeq DNA HT, 2 x 8bp, 200bp, 2 x 150 PE.
Forum: Sample Prep / Library Generation 11-23-2015, 08:23 AM
Replies: 6
Views: 2,300
Posted By Ingeneious
Strand bias in amplicon sequencing

I am seeing random strand bias in amplicon reads on an Illumina MiSeq run. Some of the amplicons have effectively complete dropout of the forward or reverse read. This occurs across every sample on...
Forum: Illumina/Solexa 09-28-2015, 11:19 AM
Replies: 3
Views: 3,618
Posted By Ingeneious
Did you add any kind of termination to the...

Did you add any kind of termination to the blocking oligo?
Forum: Illumina/Solexa 09-15-2015, 09:09 AM
Replies: 4
Views: 1,667
Posted By Ingeneious
MiSeq PM plan required?

Hi all,
We are trying to decide between Illumina's maintenance plans for our sequencers and wondering what everyone thinks of the basic vs comprehensive plan. Here is Illumina's comparison data...
Forum: Sample Prep / Library Generation 06-19-2015, 04:58 AM
Replies: 17
Views: 15,475
Posted By Ingeneious
So same as BA and TapeStation + 20min prep....

So same as BA and TapeStation + 20min prep. What's the per sample cost of the LapChip GX with reagents/chips?
Forum: Sample Prep / Library Generation 06-18-2015, 09:34 PM
Replies: 17
Views: 15,475
Posted By Ingeneious
Yeah, my boss said a new one was coming out that...

Yeah, my boss said a new one was coming out that would do 96 samples. Good to hear details.
Forum: Sample Prep / Library Generation 06-18-2015, 09:29 PM
Replies: 15
Views: 21,377
Posted By Ingeneious
That is what I am thinking, too, but I have never...

That is what I am thinking, too, but I have never seen a library prep protocol using it. Everyone seems to be using Klenow 3 - 5 exo for A-tailing and I have seen various combinations of enzymes for...
Forum: Sample Prep / Library Generation 06-18-2015, 11:11 AM
Replies: 15
Views: 21,377
Posted By Ingeneious
End Repair + A-Tailing - how do they do it?

Some of the popular library prep kits out there (i.e. NEBNext Ultra and KAPA Hyper) are able to combine end repair and A-tailing into one step. I would like to do the same with minimal cost, but am...
Forum: Sample Prep / Library Generation 06-18-2015, 10:39 AM
Replies: 17
Views: 15,475
Posted By Ingeneious
I have used the BA a lot in the past and recently...

I have used the BA a lot in the past and recently switched to the TapeStation. For convenience, it is pretty simple and accuracy is good enough to use for NGS library prep. My previous lab was using...
Forum: Sample Prep / Library Generation 06-10-2015, 04:41 PM
Replies: 0
Views: 907
Posted By Ingeneious
[delete]

Please delete. I found my answer.
Forum: Illumina/Solexa 05-21-2015, 06:48 PM
Replies: 4
Views: 2,806
Posted By Ingeneious
What kind of values have you seen with say a full...

What kind of values have you seen with say a full TruSeq adapter?

That's a good idea. I tried something similar by comparing the annealed(?) product versus an equimolar mixture of unannealed...
Forum: Illumina/Solexa 05-21-2015, 08:06 AM
Replies: 4
Views: 2,806
Posted By Ingeneious
Custom Index Adapters

How would I verify whether custom adapters have annealed properly? I get odd results when trying to quantify using QuBit, PicoGreen or TapeStation. Since the majority of the adapter is still single...
Forum: Illumina/Solexa 04-08-2015, 01:03 PM
Replies: 2
Views: 3,388
Posted By Ingeneious
Our FAS said they were moving the indexing...

Our FAS said they were moving the indexing primers onboard with the V2 chemistry. Before you had to directly pipette them in before each run. Maybe that is why?
Forum: Illumina/Solexa 01-27-2015, 10:32 AM
Replies: 53
Views: 85,524
Posted By Ingeneious
They had the HiScanSQ that did arrays plus...

They had the HiScanSQ that did arrays plus sequencing and that was quickly discontinued. Asked an Illumina employee about it and he said it seemed like they were trying to make peanut butter and...
Forum: Illumina/Solexa 01-19-2015, 11:50 AM
Replies: 145
Views: 319,578
Posted By Ingeneious
I think your index in the 3' to 5' read was off...

I think your index in the 3' to 5' read was off for so I adjusted it. Based on the 07-14 Illumina Customer letter for TruSeq HT indexes, the dual index would look something like this?

5'...
Forum: Illumina/Solexa 01-19-2015, 10:34 AM
Replies: 4
Views: 4,935
Posted By Ingeneious
Thanks, this makes sense. According to the paper,...

Thanks, this makes sense. According to the paper, it looks like contamination is also an issue, if you pool after PCR.
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