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Search: Posts Made By: luc
Forum: Introductions 11-07-2019, 05:29 PM
Replies: 1
Views: 159
Posted By luc
Welcome Saba! ...

Welcome Saba!
http://kellis.blogs.ccps.us/files/2016/08/cropped-cropped-scientist-clipart-science-clip-art-school1.png
Forum: Sample Prep / Library Generation 11-07-2019, 05:24 PM
Replies: 1
Views: 189
Posted By luc
Not a good explanation, nevertheless: perhaps...

Not a good explanation, nevertheless: perhaps your stock of index primers is degrading or got contaminated? You might want to order a few fresh oligos and compare?
Forum: Illumina/Solexa 11-05-2019, 09:02 AM
Replies: 18
Views: 465
Posted By luc
This is using custom sequencing primers? The...

This is using custom sequencing primers?

The heating and cooling elements are not calibrated exactly the same for all MiSeqs (by Illumina). Some risky custom sequencing primer designs can work...
Forum: Introductions 11-04-2019, 08:18 PM
Replies: 1
Views: 180
Posted By luc
Well ........ welcome Tom! ...

Well ........ welcome Tom!

https://images-na.ssl-images-amazon.com/images/S/sgp-catalog-images/region_US/wb-883316329542-Full-Image_GalleryBackground-en-US-1484348613962._SX1080_.jpg
Forum: Sample Prep / Library Generation 10-30-2019, 04:34 PM
Replies: 2
Views: 242
Posted By luc
Yep, bead cleanups will never be perfect. You can...

Yep, bead cleanups will never be perfect. You can only enrich for molecules of the desired size range.
Many protocols suggest two rounds of bead cleanups likey to safeguard for less than optimal...
Forum: Sample Prep / Library Generation 10-08-2019, 06:18 PM
Replies: 10
Views: 627
Posted By luc
It will. Who says molecular...

It will.

Who says molecular physics/chemistry is easy? These signals are likely due to the nature of dyes. Likely the HS-DNA dye will mostly bind to dsDNA to become fluorescent, but also to...
Forum: Sample Prep / Library Generation 10-03-2019, 12:08 PM
Replies: 10
Views: 627
Posted By luc
I guess your RNA coul actually be clean, DNA-free.

I guess your RNA coul actually be clean, DNA-free.
Forum: Sample Prep / Library Generation 10-03-2019, 09:21 AM
Replies: 10
Views: 627
Posted By luc
Qubit DNA assays will also measure Doublestranded...

Qubit DNA assays will also measure Doublestranded RNA, e.g. ds parts of rRNA.
Do you see any signs of DNA on the Bioanalyzer?
Forum: Sample Prep / Library Generation 09-11-2019, 05:19 PM
Replies: 1
Views: 593
Posted By luc
I am not aware of such a protocol. Best sequence...

I am not aware of such a protocol. Best sequence the library to low coverage as a spike-in into another run first - using different barcodes obviously.
Forum: Sample Prep / Library Generation 09-11-2019, 05:14 PM
Replies: 5
Views: 778
Posted By luc
Very, very likely your libraries will be...

Very, very likely your libraries will be perfectly fine. 45C should not be a problem, even in pure H2O. This is my guess, though.
Forum: Sample Prep / Library Generation 09-09-2019, 04:37 PM
Replies: 5
Views: 778
Posted By luc
I believe this recommendation usually applies to...

I believe this recommendation usually applies to RNA samples - which are more heat sensitive.

Depending on the buffer (some labs might use be pure H2O) and the lengths of fragments, using low...
Forum: Sample Prep / Library Generation 09-05-2019, 10:14 AM
Replies: 1
Views: 624
Posted By luc
What type of size selection did you use? I...

What type of size selection did you use?
I guess, these results are likely due to the lack of size-selection and low inputs? since you were working low integrity/quality RNA samples from fixed...
Forum: Sample Prep / Library Generation 09-03-2019, 04:29 PM
Replies: 1
Views: 521
Posted By luc
I had not seen these before. The idea looks very...

I had not seen these before. The idea looks very interesting.

I would assume the XNA clamps are added to the PCR amplification and that they are universal for the TruSeq adapter sequences. Best...
Forum: Sample Prep / Library Generation 08-15-2019, 05:53 PM
Replies: 2
Views: 762
Posted By luc
What type of libraries are you generating? How...

What type of libraries are you generating? How are you measuring concentrations?

I would suggest to avoid Phusion for complex libraries. It is fine for amplicon sequencing. Phusion has been shown...
Forum: General 08-15-2019, 05:13 PM
Replies: 1
Views: 1,143
Posted By luc
It is not necessary to bring all libraries to the...

It is not necessary to bring all libraries to the same concentration before pooling - although many labs do it that way.
You could also pool equal amounts of each library (e.g. same number of...
Forum: Sample Prep / Library Generation 08-13-2019, 09:32 AM
Replies: 1
Views: 549
Posted By luc
In our experience Quantseq seems to perform...

In our experience Quantseq seems to perform better than other 3' tag-seq methods that rely on tagmentation for adding the 5' adapter. We do not have hard data, though.
Forum: Bioinformatics 08-08-2019, 10:15 PM
Replies: 10
Views: 1,227
Posted By luc
Perhaps there just was a mistake/typo during the...

Perhaps there just was a mistake/typo during the demultiplexing/bcl2fastq conversion. Just talk to your facility.
Forum: Illumina/Solexa 07-22-2019, 02:13 PM
Replies: 3
Views: 810
Posted By luc
Sorry, I do not fully understand your description...

Sorry, I do not fully understand your description of the problem. In any case, it would be best to pool your libraries at a low percentage with other (best complex) libraries (that have differing...
Forum: Sample Prep / Library Generation 07-15-2019, 11:33 PM
Replies: 6
Views: 783
Posted By luc
search Ebay for "Magnetic Bead Separation Rack...

search Ebay for "Magnetic Bead Separation Rack for 96-Well PCR Tube Plate AMPure illumina"
or
"Magnetic beads separator rack for Ampure beads made for 0.3 mL PCR tubes", $29,
or "Magnetic Beads...
Forum: Sample Prep / Library Generation 07-15-2019, 11:09 AM
Replies: 6
Views: 783
Posted By luc
The beads should be cheaper than the columns, ...

The beads should be cheaper than the columns, you do not need to use the m most expensive ones (Ampure). You can get also cheap magnet setups from ebay.

Zymo has columns for size selection, I...
Forum: Sample Prep / Library Generation 06-19-2019, 05:27 PM
Replies: 2
Views: 1,515
Posted By luc
We have not tried this protocol. One question is:...

We have not tried this protocol. One question is: are your cells in a buffer very low in metals like Mg++ ? The protocol suggests a 1:500 dilution of Phusion HF buffer (presumably 1:500 of a 1x...
Forum: Illumina/Solexa 06-15-2019, 11:10 AM
Replies: 2
Views: 1,154
Posted By luc
No compatibility problems at all.

No compatibility problems at all.
Forum: Sample Prep / Library Generation 06-12-2019, 05:51 PM
Replies: 9
Views: 5,321
Posted By luc
Covaris does now sell plastic plates and strips...

Covaris does now sell plastic plates and strips for shearing on their very latest sonicators:
https://covaris.com/products/afa-tubes-and-vials/afa-tube/
They are a tiny bit cheaper.



You...
Forum: Bioinformatics 05-28-2019, 10:23 AM
Replies: 2
Views: 702
Posted By luc
??? UMIs have been used since at least...

???

UMIs have been used since at least 2013, then sequenced on an old GAII:
"Exploring local immunological adaptation of two stickleback ecotypes by experimental infection and transcriptome-wide...
Forum: Illumina/Solexa 05-24-2019, 06:25 PM
Replies: 11
Views: 1,488
Posted By luc
Illumina did upgrade the HS2500 sequencers of a...

Illumina did upgrade the HS2500 sequencers of a few selected companies to V4 chemistry compatibility - overall very few such compatible machines did exist. With this chemistry however, the HS2500 is...
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