SEQanswers

Go Back   SEQanswers > Search Forums


Showing results 1 to 25 of 55
Search took 0.00 seconds.
Search: Posts Made By: LVAndrews
Forum: Sample Prep / Library Generation 10-18-2019, 03:42 PM
Replies: 6
Views: 1,473
Posted By LVAndrews
The Tween modification plus 6kV for 15sec...

The Tween modification plus 6kV for 15sec injection on the RNA kit has yielded perfect data for the last 4 injections. I am once again satisfied with this instrument, though Agilent tech support was...
Forum: Sample Prep / Library Generation 10-18-2019, 11:25 AM
Replies: 6
Views: 1,473
Posted By LVAndrews
Ran the RNA kit (DNF-471) with an injection of...

Ran the RNA kit (DNF-471) with an injection of 5.5kV for 10sec, and everything worked well. These were samples that had been run previously, and I only loaded 1uL per sample while also bringing the...
Forum: Sample Prep / Library Generation 10-16-2019, 05:12 PM
Replies: 6
Views: 1,473
Posted By LVAndrews
Today I modified the electrophoresis conditions,...

Today I modified the electrophoresis conditions, specifically the injection component. Our software has the injection occur for 5 seconds at 3kV for the standard sensitivity NGS kit (DNF-473). I...
Forum: Sample Prep / Library Generation 10-16-2019, 11:06 AM
Replies: 6
Views: 1,473
Posted By LVAndrews
@weigrc doesn't seem to matter what we run, we...

@weigrc doesn't seem to matter what we run, we just have inconsistent results. The latest saga involves both RAD libraries that were Pippin selected to 200-550nt and raw RNA extracts from cell...
Forum: Sample Prep / Library Generation 10-15-2019, 01:02 PM
Replies: 6
Views: 1,473
Posted By LVAndrews
Fragment Analyzer reliability issues

We acquired our Fragment Analyzer from AATI in March 2018. We primarily use it for NGS library QC (sizing) and to collect RIN values for RNA extracts. At first it was splendid, but within a few...
Forum: Illumina/Solexa 11-02-2016, 10:55 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
Once you have sequenced through adapters, the...

Once you have sequenced through adapters, the garbage sequence that is produced afterward has very low q-scores. If you have adapter/primer sequences that are ~60mer and you are sequencing on a 2x250...
Forum: Illumina/Solexa 10-27-2016, 11:03 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
I don't know of a pub describing this...

I don't know of a pub describing this jackstraw/birds nesting effect, but there was a nice pub last month showing the effect of high cycling conditions on chimera formation...
Forum: Illumina/Solexa 10-24-2016, 05:02 PM
Replies: 44
Views: 19,824
Posted By LVAndrews
Phasing/prephasing are measures of signal purity....

Phasing/prephasing are measures of signal purity. Ideally they are zero, but that will never happen. It is an estimate more or less of the ratio of fragments per cluster that are either gaining an...
Forum: Illumina/Solexa 10-22-2016, 11:52 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
When I run those primers for 16S v4 I load at 8pM...

When I run those primers for 16S v4 I load at 8pM with 30% PhiX. Of course the Illumina people will tell you you are wasting reads with the extra phix, but I worry a lot about cluster mixing,...
Forum: Illumina/Solexa 10-22-2016, 11:09 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
1100 may be high. Just watch your...

1100 may be high. Just watch your phasing/prephasing. With long inserts you could see loss of data after turn-around.

I ran some ITS inserts recently that had full construct lengths ~650bp with...
Forum: Illumina/Solexa 08-22-2016, 10:14 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
As your insert size increases, you must also...

As your insert size increases, you must also decrease load concentration. For fungal ITS2 or v4-v5 (515-926) inserts I load at 8pM total with the same 30% PhiX.

My understanding is that 12.5pM is...
Forum: Illumina/Solexa 08-19-2016, 10:51 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
Your math is off. 12.5pM PhiX on a v2 kit should...

Your math is off. 12.5pM PhiX on a v2 kit should yield density of PhiX alone in the 850-1000k/mm range. Your total loading concentration if your quantification is correct will have been 16pM (78%...
Forum: Bioinformatics 07-06-2016, 10:43 AM
Replies: 8
Views: 4,040
Posted By LVAndrews
fastq-join works OK, you just need to relax it...

fastq-join works OK, you just need to relax it because it is so stringent. I use -m 30 -p 30 with good success. Some data is crappier than other data and may need additional grooming before it can be...
Forum: Illumina/Solexa 12-15-2014, 03:25 PM
Replies: 55
Views: 31,338
Posted By LVAndrews
I have run the 515/806 products on 300, 500, and...

I have run the 515/806 products on 300, 500, and 600 cycle kits. It is no different, but as jdelton says, read 2 in the 600 cycle kits is noisy. Schloss posted some data somewhere that he tried...
Forum: Illumina/Solexa 12-15-2014, 03:16 PM
Replies: 55
Views: 31,338
Posted By LVAndrews
You probably already figured this out, but I just...

You probably already figured this out, but I just use a 10uL pipette. Bubbles aren't going to make a bit of difference here. Pipette the primer to the side of the well where you can see it. Then...
Forum: Bioinformatics 06-03-2014, 03:11 PM
Replies: 23
Views: 5,617
Posted By LVAndrews
Has anyone had success with command-line isaac? ...

Has anyone had success with command-line isaac? I have a non-model organism I would like to push through this pipeline (ponderosa pine). If I make success, will try to post some commands here for...
Forum: Illumina/Solexa 04-04-2014, 05:12 PM
Replies: 44
Views: 19,824
Posted By LVAndrews
You are right about the genome-size concern in...

You are right about the genome-size concern in terms of transposase efficiency, but in terms of fragment size, the main factor will be how intact your DNA truly is.

I've never heard the term bird...
Forum: Illumina/Solexa 04-03-2014, 05:50 PM
Replies: 44
Views: 19,824
Posted By LVAndrews
Forgot to add why I replied in the first place. ...

Forgot to add why I replied in the first place. We order all of our own indexed primers and adapters from our synthesis source. Way cheaper than getting them as a kit. Simple desalted oligos are...
Forum: Illumina/Solexa 04-03-2014, 05:47 PM
Replies: 44
Views: 19,824
Posted By LVAndrews
First, I think I said something slightly...

First, I think I said something slightly incorrectly. I didn't mean to say genome-size dependent, I meant "intact" DNA size dependent. Decent quality DNA extractions yield DNA sheared to around...
Forum: Illumina/Solexa 04-03-2014, 09:33 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
1) I have. It works. Also dependent on genome...

1) I have. It works. Also dependent on genome size.

2) Illumina's recommendation is exactly a 0.5X cleanup. You do incur loss of target DNA at this concentration. Reducing to 0.4X, you could...
Forum: Illumina/Solexa 04-02-2014, 06:45 PM
Replies: 44
Views: 19,824
Posted By LVAndrews
M4TTN: Results looked splendid. Output as...

M4TTN: Results looked splendid. Output as anticipated (17G), ~80%>q30.

I hadn't seen mcnelson's post despite having posted in this thread myself previously, but we also have abandoned the bead...
Forum: Illumina/Solexa 03-28-2014, 10:53 AM
Replies: 44
Views: 19,824
Posted By LVAndrews
austinso, Yaximiks recipe does diulte 100fold...

austinso, Yaximiks recipe does diulte 100fold with hyb buffer. Since he(?) does 3ul NaOH and 3ul of pool then the final dilution achieves this.

Thanks rnaeye for the clarification. I almost...
Forum: Illumina/Solexa 03-27-2014, 02:20 PM
Replies: 44
Views: 19,824
Posted By LVAndrews
Doing it!! My difficult library is 2.7nM. Too...

Doing it!! My difficult library is 2.7nM. Too lazy to bead-concentrate and re-quant. Plus I like to try useful mods. Loading a 600 cycle kit now. Thanks!!
Forum: Illumina/Solexa 03-27-2014, 01:30 PM
Replies: 44
Views: 19,824
Posted By LVAndrews
Yaximik, I am suddenly finding myself interested...

Yaximik, I am suddenly finding myself interested in your acetate mod. I have some libraries that just don't want to reach 4nM. Your mod suggests adequate clustering can be had with lower...
Forum: Bioinformatics 01-20-2014, 09:18 AM
Replies: 4
Views: 1,937
Posted By LVAndrews
I thought I had tried that previously, but the...

I thought I had tried that previously, but the user went and tried the free version of Geneious and used that instead (pretty great assembler). Their lab wound up purchasing a license as did we. If...
Showing results 1 to 25 of 55

 


All times are GMT -8. The time now is 05:47 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO