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Search: Posts Made By: bunce
Forum: Illumina/Solexa 06-06-2016, 10:41 PM
Replies: 13
Views: 4,218
Posted By bunce
Hi Biokiwi.... it kind of depends on how long...

Hi Biokiwi.... it kind of depends on how long your amplicon is.

We do a fair bit of single-end sequencing (amplicons ~200bp). Here the setup is (post ligation....
Forum: Illumina/Solexa 06-06-2016, 05:59 PM
Replies: 13
Views: 4,218
Posted By bunce
Hi BioKiwi - if your indexes are read 'in-line'...

Hi BioKiwi - if your indexes are read 'in-line' then (separate) indexing reads are not needed. For most of our amplicon workflows we do not carry out indexing, instead we deconvolute the data based...
Forum: Illumina/Solexa 05-30-2016, 03:51 PM
Replies: 5
Views: 2,054
Posted By bunce
Hi Maddoc1ck - we found this protocol helpful...

Hi Maddoc1ck - we found this protocol helpful when using a 'home-made' PCR free approach. http://www.ncbi.nlm.nih.gov/pubmed/21431776

Good luck with your workflows.
Forum: Illumina/Solexa 02-03-2016, 03:04 AM
Replies: 2
Views: 1,244
Posted By bunce
Hi francesca3877, There are many, many...

Hi francesca3877,

There are many, many reason for a poor sequencing run - the #1 cause is usually clustering density (are using using v2 or V3 chemistry). You can't just look at the clustering...
Forum: Illumina/Solexa 01-29-2016, 08:41 PM
Replies: 22
Views: 4,796
Posted By bunce
This is an interesting thread - we also had a...

This is an interesting thread - we also had a drop in MiSeq performance (in low complexity libraries) after upgrading to 2.6 and like some of you rolled back to 2.5. I was told there was nothing in...
Forum: Illumina/Solexa 01-18-2016, 01:33 AM
Replies: 5
Views: 2,338
Posted By bunce
Hi AnnaMC, Reading melt curves without all the...

Hi AnnaMC,
Reading melt curves without all the information is a bit like reading tea leaves but I will have a shot ;-)

Is there a no template control in here? I ask as this is the most obvious...
Forum: Illumina/Solexa 12-16-2015, 01:51 PM
Replies: 13
Views: 4,218
Posted By bunce
Hi Thermophile, #1) In the adaptors we use the...

Hi Thermophile, #1) In the adaptors we use the Nextera sequences - so yes from memory we substituted out the old PE one when designing this workflow (I would need to do some digging to be compleltely...
Forum: Illumina/Solexa 12-10-2015, 12:24 PM
Replies: 8
Views: 3,244
Posted By bunce
Hi SunPenguin. I agree with Jessica it is likely...

Hi SunPenguin. I agree with Jessica it is likely a low complexity issue - but would add that it might be exacerbated by densities above 900k. We have run a lot of amplicon low compexity libraries and...
Forum: Illumina/Solexa 11-24-2015, 10:08 PM
Replies: 2
Views: 2,465
Posted By bunce
Hi discoveradnan, you are correct that these are...

Hi discoveradnan, you are correct that these are 'common sequences' but are missing the fact that in the second round of PCR the primer binds to (and extends off) to opposite strand (i.e not the...
Forum: Illumina/Solexa 11-11-2015, 02:26 PM
Replies: 4
Views: 1,332
Posted By bunce
Hi Xaki. Yes - clusters formed. I am not sure why...

Hi Xaki. Yes - clusters formed. I am not sure why we could never get decent sequences. I suspect it might be that the 'vision' that all the libraries are lined up as nice organised vertical strands...
Forum: Illumina/Solexa 11-11-2015, 03:15 AM
Replies: 4
Views: 1,332
Posted By bunce
Hi Xaki, yes we tried a complete overlap with no...

Hi Xaki, yes we tried a complete overlap with no success. Still not 100% why this didn't work but we gave up after about 3-4 runs (and tried a lot of things). So we ended up using a custom primer....
Forum: Illumina/Solexa 10-28-2015, 02:21 PM
Replies: 24
Views: 8,259
Posted By bunce
You might want to call Illumina and get some...

You might want to call Illumina and get some formal help. On the two occasions that we have had a premature run termination (primarily when the run hits the 'end of a amplicon' and the signal falls...
Forum: Illumina/Solexa 10-28-2015, 07:58 AM
Replies: 24
Views: 8,259
Posted By bunce
Hi GA-J, Are you sequencing amplicons? - you can...

Hi GA-J, Are you sequencing amplicons? - you can attempt to salvage the data by altering the sample sheet to do 300 in read 1 and then~240 for R2 (you do this in MiSeq reporter -...
Forum: Illumina/Solexa 10-21-2015, 05:21 AM
Replies: 2
Views: 1,414
Posted By bunce
Hi jsvinco, Each kit is loaded with 25...

Hi jsvinco,
Each kit is loaded with 25 additional cycles for indexing. So unless you need both indexing reads the answer is a 'yes'. If you need both indexing reads as well as the 50 cycles you...
Forum: Illumina/Solexa 09-22-2015, 03:32 PM
Replies: 6
Views: 2,741
Posted By bunce
Hi - never had the "out of spec" error after ~100...

Hi - never had the "out of spec" error after ~100 miseq runs. As cheezemeister mentioned the only time we have had focus issues is due to under clustering - when we were dialing-in our quant. We also...
Forum: Illumina/Solexa 09-15-2015, 03:50 PM
Replies: 4
Views: 2,443
Posted By bunce
Hi Urchin - drop me an email if you want to...

Hi Urchin - drop me an email if you want to discuss any of this. Contamination in NGS workflows (especially involving amplicons) is, as you point out, something you want to get right from the outset....
Forum: Illumina/Solexa 09-15-2015, 03:36 PM
Replies: 4
Views: 2,443
Posted By bunce
Hi Urchin, In my experience of 'minimising'...

Hi Urchin, In my experience of 'minimising' contamination/artefacts the workflow (as opposed to the equipment) is the thing to get right - The best advice I could give you here is to:
1) Separate...
Forum: Illumina/Solexa 06-14-2015, 12:38 AM
Replies: 12
Views: 3,709
Posted By bunce
Hi All, I have seen this only once in 100 MiSeq...

Hi All,
I have seen this only once in 100 MiSeq runs - I was told... " xxxx asked me to get in touch with you regarding washing the MiSeq after bright spots have been seen. We don't have a...
Forum: Illumina/Solexa 05-28-2015, 02:35 AM
Replies: 10
Views: 3,173
Posted By bunce
Hi Jon, It might be OK to run this with such low...

Hi Jon, It might be OK to run this with such low diversity - but I would not risk it. If it were me I would take a previous dual-indexed library (with different indexes to those in your library) and...
Forum: Illumina/Solexa 05-05-2015, 06:18 PM
Replies: 15
Views: 4,014
Posted By bunce
Hi Kurt, Not sure if this will help but here is...

Hi Kurt,
Not sure if this will help but here is my 2cents worth.... The entire ligation reaction will be inefficient, most of the template will not ligate 'correctly'. The proportion you do get to...
Forum: Illumina/Solexa 04-17-2015, 11:15 PM
Replies: 13
Views: 4,218
Posted By bunce
Hi kmcarr - thanks of the heads-up have corrected...

Hi kmcarr - thanks of the heads-up have corrected them (they used to work!) - the elipes are a truncation that SeqAnswers uses - the refs are;
1)Phillipe et al 2015. Nucleic Acids Research....
Forum: Illumina/Solexa 04-17-2015, 05:34 PM
Replies: 13
Views: 4,218
Posted By bunce
as for tips - we generate 1-2ug of amplicon...

as for tips - we generate 1-2ug of amplicon (crude calculation by nano drop) , ligate adapters at a ratio of 3:1 (adapter:amplicon).... we then Pippin Prep the library as the biggest hassle is...
Forum: Illumina/Solexa 04-15-2015, 08:10 PM
Replies: 3
Views: 1,803
Posted By bunce
Hi - I agree with Thermophile. One of the...

Hi - I agree with Thermophile. One of the simplest things you can do is an in silico analysis of the binding sites in your bacterial targets for the V3-V4 primers. Over a number of PCR cycles even...
Forum: Illumina/Solexa 04-13-2015, 11:59 PM
Replies: 13
Views: 4,218
Posted By bunce
Hi Thermophile, if you have to go down the 2-step...

Hi Thermophile, if you have to go down the 2-step PCR approach then consider the implications (for indexing) in these two papers;
...
Forum: Illumina/Solexa 04-08-2015, 10:35 PM
Replies: 2
Views: 2,816
Posted By bunce
I was told something similar - there is a change...

I was told something similar - there is a change in the index/custom primer setup from V1 to V2.
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