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Forum: Sample Prep / Library Generation 08-15-2019, 04:53 PM
Replies: 2
Views: 253
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, 04:13 PM
Replies: 1
Views: 241
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, 08:32 AM
Replies: 1
Views: 120
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, 09:15 PM
Replies: 10
Views: 779
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, 01:13 PM
Replies: 3
Views: 365
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, 10:33 PM
Replies: 6
Views: 412
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, 10:09 AM
Replies: 6
Views: 412
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, 04:27 PM
Replies: 2
Views: 1,300
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, 10:10 AM
Replies: 2
Views: 691
Posted By luc
No compatibility problems at all.

No compatibility problems at all.
Forum: Sample Prep / Library Generation 06-12-2019, 04:51 PM
Replies: 9
Views: 5,006
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, 09:23 AM
Replies: 2
Views: 513
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, 05:25 PM
Replies: 11
Views: 1,124
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...
Forum: Illumina/Solexa 05-24-2019, 12:51 PM
Replies: 11
Views: 1,124
Posted By luc
Very likely yes, since Illumina is manipulating...

Very likely yes, since Illumina is manipulating the prices to their liking. HS4K reagent costs did increase by 6% this year (everything else by 3%); the costs for the small NovaSeq flowcells OTOH...
Forum: General 05-23-2019, 06:30 PM
Replies: 6
Views: 1,081
Posted By luc
You will need a magnetic bead separation plate to...

You will need a magnetic bead separation plate to use the beads for cleanups in 96-well PCR plates.

Zymo has DNA clean & concentrate kits in plate format (silica columns). You will require a...
Forum: Illumina/Solexa 05-22-2019, 04:52 PM
Replies: 3
Views: 744
Posted By luc
I would check out the SMART-SEQ2 protocol. They...

I would check out the SMART-SEQ2 protocol. They did homebrew something similar. I vaguely remember SDS to denature the enzyme.
Forum: Illumina/Solexa 05-22-2019, 04:38 PM
Replies: 11
Views: 1,124
Posted By luc
I guess it is not the throughput nor the lifespan...

I guess it is not the throughput nor the lifespan of the instruments that kills the older sequencers; it is mostly the comparatively high costs per base. The exception are the MiSeqs because Illumina...
Forum: General 05-21-2019, 08:43 PM
Replies: 3
Views: 1,016
Posted By luc
The presence of a prominent short fragment band...

The presence of a prominent short fragment band indicates that degradation is not the problem. Bacterial RNA-seq obviously will not make use of poly-A enrichment and thus is not very sensitive to...
Forum: Sample Prep / Library Generation 05-14-2019, 07:29 PM
Replies: 6
Views: 831
Posted By luc
I do not believe that the bead surface is...

I do not believe that the bead surface is strongly limiting the DNA amounts that can be precipitated onto it - at least not when using standard Ampure (PEG/NaCl) buffer.
It is worth a try.
Forum: Sample Prep / Library Generation 05-10-2019, 06:20 AM
Replies: 2
Views: 775
Posted By luc
What QC information do you have for the libraries...

What QC information do you have for the libraries before pooling and capture?
After sequencing, how is the read number within the pools?
Forum: Introductions 05-07-2019, 06:54 AM
Replies: 2
Views: 659
Posted By luc
Welcome to the forum! But better ask such...

Welcome to the forum!
But better ask such questions in the library generation forum.
That said I can't imagine that this would generate usable data - I assume the fixation is too strong in FFPE...
Forum: Illumina/Solexa 05-06-2019, 04:58 PM
Replies: 10
Views: 1,289
Posted By luc
I am just speculating. AFAIU, the remaining...

I am just speculating. AFAIU, the remaining oligos will be kicked off by nick-translation/strand-displacement in addition to the gap filling; I assume there is no ligation happening. (Perhaps the...
Forum: Illumina/Solexa 04-26-2019, 09:54 AM
Replies: 3
Views: 1,341
Posted By luc
Every library molecule and every strand has both...

Every library molecule and every strand has both the P5 and P7 sequences.
Only one of the ends hybridizes initially, because of the direction/complementarity of the flowcell oligos.
Forum: Illumina/Solexa 04-22-2019, 02:12 PM
Replies: 2
Views: 527
Posted By luc
PCR inhibitors?

PCR inhibitors?
Forum: Sample Prep / Library Generation 04-19-2019, 04:45 PM
Replies: 6
Views: 1,139
Posted By luc
Sorry, I have no clue what could have gone wrong....

Sorry, I have no clue what could have gone wrong. We do see significantly higher yields with the same Kapa kit using Biooscientific adapters after 7 PCR cycles.
Did you run a two-sided size...
Forum: Illumina/Solexa 04-16-2019, 06:19 PM
Replies: 2
Views: 525
Posted By luc
PCR cleanup should be comparatively "easy" and...

PCR cleanup should be comparatively "easy" and all the usual manufacturers should have good options.
Qiagen QIAquick PCR or Zymo DNA clean would be some we have used.
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