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Forum: Sample Prep / Library Generation 11-06-2018, 06:25 AM
Replies: 3
Views: 264
Posted By Genetic Librarian
Totally fine. If I remember correctly, the Qubit...

Totally fine. If I remember correctly, the Qubit kits (incl. the standards) are shipped at RT.
Forum: Sample Prep / Library Generation 07-17-2018, 06:49 AM
Replies: 6
Views: 1,137
Posted By Genetic Librarian
If you use an amplicon approach (16S v3v4) you do...

If you use an amplicon approach (16S v3v4) you do not need any form of depletion, because you just amplify bacterial 16S sequences ;). The primers are specific, so that it does not matter how much of...
Forum: Sample Prep / Library Generation 07-17-2018, 06:19 AM
Replies: 6
Views: 1,137
Posted By Genetic Librarian
No, the process is much simpler as you think. You...

No, the process is much simpler as you think. You simply isolate total RNA and reverse transcribe that into cDNA using random primers. Than you do your normal 16S amplicon workflow on that cDNA. You...
Forum: Sample Prep / Library Generation 07-16-2018, 11:51 PM
Replies: 6
Views: 1,137
Posted By Genetic Librarian
Well, on DNA level you get everything, dead or...

Well, on DNA level you get everything, dead or alive. On RNA level, you get the viable bacterial community, which can be very different in sites of active immune response for example.
Forum: Illumina/Solexa 04-25-2018, 11:52 PM
Replies: 7
Views: 1,461
Posted By Genetic Librarian
You also have to keep in mind that the flowcells...

You also have to keep in mind that the flowcells and clustering methods are not the same in the NovaSeq as in the MiSeq / NextSeq. A patterned flowcell is much worse for sequencing long libraries,...
Forum: Sample Prep / Library Generation 04-03-2018, 01:56 AM
Replies: 5
Views: 1,479
Posted By Genetic Librarian
Just to make sure you are aware: The Tn5 does...

Just to make sure you are aware:
The Tn5 does not only fragment your DNA, it also adds primer binding sites that are essential for the downstream Nextera workflow. In your post, it sounds as if you...
Forum: Illumina/Solexa 12-19-2017, 12:44 AM
Replies: 2
Views: 872
Posted By Genetic Librarian
Data Output of Sequencer divided by (target size...

Data Output of Sequencer divided by (target size * coverage) = number of samples.

12 samples in your case not accounting for off-target reads (phiX, enrichments etc.)
Forum: Illumina/Solexa 12-08-2017, 04:28 AM
Replies: 9
Views: 977
Posted By Genetic Librarian
During the denature and dilute process you dilute...

During the denature and dilute process you dilute your library to the final loading concentration with the provided buffer based on your final Qubit quantification of the library pool. So, instead of...
Forum: Illumina/Solexa 11-22-2017, 11:00 PM
Replies: 9
Views: 1,222
Posted By Genetic Librarian
That should not be the case, because you pool...

That should not be the case, because you pool equimolar.
Forum: Illumina/Solexa 11-22-2017, 01:10 AM
Replies: 9
Views: 1,222
Posted By Genetic Librarian
Insert size is a problem because bias works in...

Insert size is a problem because bias works in favour of smaller fragments.
So if you mix a 300 bp library and a 500 bp library 50/50, you will end up with a ~70/30 read distribution.
One reason is...
Forum: Illumina/Solexa 11-21-2017, 07:20 AM
Replies: 9
Views: 1,222
Posted By Genetic Librarian
well, mixing two libraries is a tricky task in...

well, mixing two libraries is a tricky task in general, no matter if PCR-free or not.
It works well with libraries of the same fragment size. If the two libraries are of different sizes, there is a...
Forum: Sample Prep / Library Generation 10-19-2017, 01:25 AM
Replies: 3
Views: 1,222
Posted By Genetic Librarian
My wild guess would be on low ligation efficacy,...

My wild guess would be on low ligation efficacy, giving the final PCR no target to amplify.
If that is true, you can check that by quantifying your library by Qubit and by qPCR (e.g. Kapa library...
Forum: Sample Prep / Library Generation 10-06-2017, 05:51 AM
Replies: 7
Views: 3,547
Posted By Genetic Librarian
Prices are already up on their website. Per...

Prices are already up on their website.
Per sample it is more expensive than Nextera XT and TruSeq Nano, but cheaper than Nextera (at least in EURO). So if you use all three kits with bias to...
Forum: Sample Prep / Library Generation 10-06-2017, 04:38 AM
Replies: 7
Views: 3,547
Posted By Genetic Librarian
New kit: Nextera DNA-Flex

Hi,
I am sure many of you got the newsletter from Illumina today.
So, apparently the New DNA Flex kit is an all-in-one solution for Nextera XT, Nextera and TruSeq DNA Nano Users.
Main benefit...
Forum: Illumina/Solexa 09-14-2017, 11:53 PM
Replies: 6
Views: 1,156
Posted By Genetic Librarian
We always amplified the mtGenome in two long...

We always amplified the mtGenome in two long range PCRs, so we were never limited in material.
At the end, our final libraries were quite large (fragment size) and output was good, so I guess it...
Forum: Illumina/Solexa 09-14-2017, 11:45 PM
Replies: 6
Views: 1,156
Posted By Genetic Librarian
Hi, Question 1: Your output requirement is...

Hi,

Question 1: Your output requirement is 16kb (target size) * 3000 (coverage)

Thus, per sample you would need 48 Megabases as output.
A MiSeq v2 2x 150 bp PE run e.g. has an output of 5...
Forum: Illumina/Solexa 09-14-2017, 12:06 AM
Replies: 1
Views: 670
Posted By Genetic Librarian
Hi Voijtech, first, I would recomment you to...

Hi Voijtech,

first, I would recomment you to read this for some more background:

https://genohub.com/next-generation-sequencing-guide/

Second: Getting good data for miRNA, rRNA and mRNA in a...
Forum: Illumina/Solexa 09-07-2017, 07:19 AM
Replies: 7
Views: 1,108
Posted By Genetic Librarian
The reported cluster density is not always...

The reported cluster density is not always reliable.
If a run is massively overclustered, the MiSeq can not distinguish single clusters and logically can not count them reliably.
Another indicator...
Forum: General 08-28-2017, 07:29 AM
Replies: 3
Views: 980
Posted By Genetic Librarian
This! Biggest mistake is too few replicates....

This!

Biggest mistake is too few replicates. Individual samples can drop out for a variety of reasons, so easily your glorious n=3 study has treatments with only 2 replicates in them. And you can...
Forum: RNA Sequencing 07-31-2017, 06:06 AM
Replies: 1
Views: 933
Posted By Genetic Librarian
Try to crunch some numbers. Assume X bacterial...

Try to crunch some numbers.
Assume X bacterial cells per host cell (e.g. 10, check literature) and then assume the RNA content of each cell (based on genome sizes).
Then assume how good your kits...
Forum: Illumina/Solexa 07-21-2017, 12:53 AM
Replies: 8
Views: 1,135
Posted By Genetic Librarian
Your samplesheet does not need to be generated by...

Your samplesheet does not need to be generated by IEM.
IEM is a nice tool, so you get the headers, parameters etc right.

But you can also easily open the resulting .csv in excel and add how many...
Forum: Illumina/Solexa 07-20-2017, 12:36 AM
Replies: 13
Views: 3,805
Posted By Genetic Librarian
There meanwhile is an alternative "SeqClin",...

There meanwhile is an alternative "SeqClin", which simply is already pre-diluted ProClin. A 1:5 dilution of SeqClin results in MW-Solution for the HiSeqs.
Forum: Sample Prep / Library Generation 07-05-2017, 11:12 PM
Replies: 3
Views: 2,002
Posted By Genetic Librarian
according to this post it should be a ratio of...

according to this post it should be a ratio of 0.5 or 0.6.

http://core-genomics.blogspot.de/2012/04/how-do-spri-beads-work.html
Forum: Illumina/Solexa 06-29-2017, 02:17 AM
Replies: 2
Views: 566
Posted By Genetic Librarian
1) Fake the ....complete.txt files (just copy an...

1) Fake the ....complete.txt files (just copy an old one and put a reasonable time stamp in there)
2) Correct the read length in the xml files (you might need to go one less than you think, because...
Forum: Illumina/Solexa 06-29-2017, 02:11 AM
Replies: 7
Views: 1,461
Posted By Genetic Librarian
we have successfully clustered 1000bp libraries...

we have successfully clustered 1000bp libraries (final size including adapters). You will end up with less cluster passing filter, but we were still within specs. On the lower end, I have no...
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