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Forum: Metagenomics 01-07-2014, 08:12 AM
Replies: 8
Views: 15,407
Posted By csquared
Very easy to use PANDASeq to assemble the paired...

Very easy to use PANDASeq to assemble the paired reads and then use the assembled file in QIIME.

If you can use MacQIIME, I have a simple python script and some BASH scripts that make processing...
Forum: Illumina/Solexa 01-14-2013, 02:39 PM
Replies: 55
Views: 32,611
Posted By csquared
It provides a "pad" for the primers to anneal for...

It provides a "pad" for the primers to anneal for the sequencing versus amplification primers. Hard to explain in perfect detail in text but if you diagram the amplicon and draw where each primer...
Forum: Illumina/Solexa 01-08-2013, 11:05 AM
Replies: 9
Views: 3,438
Posted By csquared
"resynthesis" vs "regeneration" is a fine line. ...

"resynthesis" vs "regeneration" is a fine line. More importantly, there are 15 cycles alternating between formamide and amplification mix during the turn, very similar to exactly what happens during...
Forum: Illumina/Solexa 01-03-2013, 12:23 PM
Replies: 2
Views: 2,621
Posted By csquared
The PCR primers used by Affymetrix to amplify the...

The PCR primers used by Affymetrix to amplify the mtDNA in two overlapping fragments for use on their sequencing array work great for amplification prior to library prep and sequencing. The...
Forum: Illumina/Solexa 01-03-2013, 12:17 PM
Replies: 9
Views: 3,438
Posted By csquared
If you want all of the details for the process,...

If you want all of the details for the process, the Bentley et al Nature paper from 2008 is the best resource for exactly how all of the chemistry works for paired-end sequencing.
...
Forum: Illumina/Solexa 12-04-2012, 10:53 AM
Replies: 12
Views: 10,994
Posted By csquared
The Caporaso primers are great primers and an...

The Caporaso primers are great primers and an elegant design. If you want to cover V1-V3 rather than V4, you can make an easy change to use 27F and 534R from the human microbiome project:

27F...
Forum: Illumina/Solexa 11-21-2012, 11:11 AM
Replies: 0
Views: 2,636
Posted By csquared
Two samples per HiSeq2500 flowcell

Just noticed a new item in the Illumina catalog:

CT-402-4001
TruSeq® Rapid Duo cBot™ Sample Loading Kit The TruSeq® Rapid Duo cBot™ Sample Loading Kit allows different samples to be loaded onto...
Forum: Illumina/Solexa 10-26-2012, 09:50 AM
Replies: 3
Views: 2,542
Posted By csquared
Size selection is definitely not needed by gel,...

Size selection is definitely not needed by gel, but a bead-based size selection to get rid of adaptor dimer is really helpful. A single 1x bead purification will get rid of stuff below 200nt prior...
Forum: Illumina/Solexa 08-30-2012, 01:07 PM
Replies: 9
Views: 6,133
Posted By csquared
The primers are the standard flowcell sequences. ...

The primers are the standard flowcell sequences. We actually now use the Kapa Biosystems kit. We buy it in very large bulk and the cost per library is very good. Better than building our own kit...
Forum: Illumina/Solexa 08-28-2012, 03:45 PM
Replies: 83
Views: 28,767
Posted By csquared
With the MiSeq software update, you can use a...

With the MiSeq software update, you can use a matrix from a different run, supposedly allowing low diversity/amplicon sequencing on the platform. Time will tell how effective that solution will be,...
Forum: Illumina/Solexa 08-28-2012, 03:36 PM
Replies: 9
Views: 5,024
Posted By csquared
Adaptors are a lot longer than 20 or 50nt. Are...

Adaptors are a lot longer than 20 or 50nt. Are you sure it isn't more adaptor? If it was a truseq kit, looks like you could have gone through the primer and into the index sequence, leading to some...
Forum: Illumina/Solexa 08-28-2012, 03:31 PM
Replies: 9
Views: 6,133
Posted By csquared
For library pooling, you will absolutely see the...

For library pooling, you will absolutely see the best results with real-time PCR quantitation prior to pooling. Of course it is possible to do well with a size estimation using bioanalyzer or...
Forum: Illumina/Solexa 08-24-2012, 11:54 AM
Replies: 9
Views: 6,133
Posted By csquared
Best choice will depend on how accurately you...

Best choice will depend on how accurately you pool the libraries in molar equivalents and if all libraries sequence with equal efficiency.

From a sequencing standpoint the two choices are pretty...
Forum: Illumina/Solexa 05-29-2012, 10:54 AM
Replies: 4
Views: 8,217
Posted By csquared
When are you doing the size selection step? ...

When are you doing the size selection step? After shearing or after ligation?
Forum: Illumina/Solexa 05-29-2012, 10:51 AM
Replies: 7
Views: 2,606
Posted By csquared
It's definitely above spec but also an output...

It's definitely above spec but also an output that is quite reproducible if done carefully.

That said, just be sure you don't have any alignment issues. We have had similar runs and every once...
Forum: Illumina/Solexa 01-06-2012, 12:18 PM
Replies: 2
Views: 2,345
Posted By csquared
Accurate quantitation and normalization before...

Accurate quantitation and normalization before pooling is the key.

Anything other than real-time PCR based quantiation and you will see variability from run to run, especially as you get to higher...
Forum: Illumina/Solexa 12-19-2011, 03:32 PM
Replies: 19
Views: 12,902
Posted By csquared
A bit over 1M cluster/mm2

A bit over 1M cluster/mm2
Forum: Illumina/Solexa 12-08-2011, 04:41 PM
Replies: 2
Views: 5,946
Posted By csquared
FWHM is definitely a very useful metric (except...

FWHM is definitely a very useful metric (except in the index read). We find it most useful to compare it between all 4 bases. The more even and overlapping the plots are, the better the run.

In...
Forum: Illumina/Solexa 12-08-2011, 04:26 PM
Replies: 6
Views: 3,169
Posted By csquared
The contract is probably worth it. The hourly...

The contract is probably worth it. The hourly rate for service is $300/hr with a 2-hour minimum. Add to that the $750-$1,500 travel costs that are added as a flat rate. However, those costs are...
Forum: Sample Prep / Library Generation 12-04-2011, 07:08 PM
Replies: 10
Views: 6,940
Posted By csquared
There are a few good options for sealing multiple...

There are a few good options for sealing multiple times during incubations and/or PCR. We use a Beckman Biomek deck with MJ thermocycler on deck. There is a reusable lid with a soft rubber gasket...
Forum: Illumina/Solexa 12-01-2011, 07:39 PM
Replies: 11
Views: 2,896
Posted By csquared
Completely agree but as far as inferring...

Completely agree but as far as inferring strandedness from an area of the genome with unknown/no annotation, the canonical splicing sites don't help much. For example, non-coding RNA. A standard...
Forum: Illumina/Solexa 12-01-2011, 07:20 PM
Replies: 10
Views: 23,090
Posted By csquared
I should have said that when using the TruSeq...

I should have said that when using the TruSeq indexes and you select the Single Index option in the new HCS, it will do 7 cycles of sequencing during the index read (6 to read the barcode and the 7th...
Forum: Illumina/Solexa 12-01-2011, 12:35 PM
Replies: 11
Views: 2,896
Posted By csquared
Not sure I agree. The presence of the splice...

Not sure I agree. The presence of the splice site sequence still doesn't tell you which strand the original transcript came from. Only that the read was generated from that strand. The production...
Forum: Illumina/Solexa 12-01-2011, 11:55 AM
Replies: 38
Views: 19,962
Posted By csquared
The loss of focus during read 3 is likely a...

The loss of focus during read 3 is likely a bubble from fluidics than a diversity problem. If you got that far with good PF clusters, good base quality and a good, flat FWHM metric, it isn't the...
Forum: Illumina/Solexa 12-01-2011, 11:47 AM
Replies: 10
Views: 23,090
Posted By csquared
It won't show results for old runs. Only runs...

It won't show results for old runs. Only runs that are configured as index runs using the new version of HCS to go along with the new SAV. If you are using the TruSeq indexes, you have use a 7-base...
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