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Search: Posts Made By: luc
Forum: General 03-22-2019, 09:19 AM
Replies: 3
Views: 270
Posted By luc
The recommended buffer would be EB (10 mM Tris,...

The recommended buffer would be EB (10 mM Tris, pH=8 to 8.4) or EBT (10mM TRIS, 0,1%Tween20) .
Forum: General 03-21-2019, 11:01 AM
Replies: 3
Views: 270
Posted By luc
Your sequencing company asks for way too much...

Your sequencing company asks for way too much library.

However, the amplification clearly should have worked as described. I would guess a simple mistake at the PCR setup is the most likely...
Forum: Pacific Biosciences 03-19-2019, 12:27 PM
Replies: 4
Views: 212
Posted By luc
PacBio captures videos from single fluorescent...

PacBio captures videos from single fluorescent molecules that are in a specific location only for milliseconds. Thus, they are trying to avoid any vibrations.
Forum: Bioinformatics 03-19-2019, 12:05 PM
Replies: 6
Views: 223
Posted By luc
They are paired-end? Which read length? You...

They are paired-end? Which read length?
You should be able to determine the insert sizes from the alignments (bam files).
Forum: Bioinformatics 03-18-2019, 07:33 PM
Replies: 6
Views: 223
Posted By luc
Just a guess: paired-end reads of short...

Just a guess: paired-end reads of short fragments; thus they are mostly overlapping; resulting in counts as multiples of two?
More info would help.
Forum: Pacific Biosciences 03-18-2019, 07:27 PM
Replies: 4
Views: 212
Posted By luc
among other things, laser & optics, filter,...

among other things, laser & optics, filter, cooling for the reagents and the entire interior, a liquid handling robot, an overengineered air table that runs on nitrogen - which was not really needed.
Forum: Sample Prep / Library Generation 03-15-2019, 05:47 PM
Replies: 1
Views: 242
Posted By luc
I am afraid there is not one kit that is...

I am afraid there is not one kit that is universally applicable.
ATACseq for example depends on the Nextera transposase.
ChIPseq will require adapter ligation.
Amplicon seq will vary a lot with...
Forum: RNA Sequencing 03-14-2019, 07:45 AM
Replies: 1
Views: 197
Posted By luc
I do not have any data to support my opinion. I...

I do not have any data to support my opinion.
I would go with number 1, due to the consistency of the RNA isolations - they will be all prepped at the same time by the same person. RNA is stable if...
Forum: Ion Torrent 03-13-2019, 05:38 PM
Replies: 4
Views: 1,970
Posted By luc
We recently got an OT2. So far it works for...

We recently got an OT2. So far it works for single channel pipetting.

We have problems with both of our multi-channel pipettes.
The magnetic module seems to have a design flaw - the magnet is too...
Forum: Illumina/Solexa 03-12-2019, 12:19 PM
Replies: 1
Views: 183
Posted By luc
Hi Olga, your math seems correct. Six...

Hi Olga,

your math seems correct. Six samples to sequence about 50 million library molecules each. Buenrostro et al. generally refer to paired-reads in their papers.
But you can certainly pool...
Forum: Epigenetics 03-07-2019, 05:57 PM
Replies: 16
Views: 5,874
Posted By luc
Yes, the Covaris recommendation matches our...

Yes, the Covaris recommendation matches our experience. You will likely need a longer sonication time. The sonication of already short fragments is comparatively inefficient.
You will have to try...
Forum: Pacific Biosciences 03-06-2019, 07:49 AM
Replies: 1
Views: 327
Posted By luc
The new library prep kit just became available -...

The new library prep kit just became available - it will be best to compare the data with the new kit.

V3 chemistry so far produces significant longer polymerase reads, given good DNA samples....
Forum: Illumina/Solexa 02-18-2019, 08:25 AM
Replies: 1
Views: 437
Posted By luc
The results do vary by sequencer (the Hiseq 4000...

The results do vary by sequencer (the Hiseq 4000 is not - or only minimally - bothered by dual matched indices); the NovaSeq OTOH can tank with these. Likely library insert sizes can also play a...
Forum: RNA Sequencing 02-14-2019, 10:14 PM
Replies: 1
Views: 370
Posted By luc
How do you know? I would suggest to follow the...

How do you know? I would suggest to follow the library prep protocol all the way to the end and then assess the library or lack thereof.
Forum: Illumina/Solexa 02-07-2019, 05:16 PM
Replies: 1
Views: 403
Posted By luc
PCR-free Y-adapter libraries can migrate in funky...

PCR-free Y-adapter libraries can migrate in funky ways and can hide adapter dimers. In short they are difficult to QC.
I would suggest a diagnostic PCR with a small aliquot of the libraries:...
Forum: Introductions 01-24-2019, 05:19 PM
Replies: 2
Views: 802
Posted By luc
Hi msharik, A big hello to Indore: ...

Hi msharik,

A big hello to Indore: https://en.wikipedia.org/wiki/Indore
Forum: Sample Prep / Library Generation 01-21-2019, 08:14 AM
Replies: 0
Views: 577
Posted By luc
"random" enzymatic DNA fragmentation - how does it work?

Hi All,

almost all sequencing library preparation kit providers now offer kits promising "random" enzymatic DNA fragmentation that are not based on transposon tagmentation - adapters are ligated...
Forum: Core Facilities 01-19-2019, 05:26 PM
Replies: 3
Views: 858
Posted By luc
Bukowski is correct. RNA-seq library prep...

Bukowski is correct. RNA-seq library prep protocols (starting from RNA) are optimized for consistency to allow quantitative DGE analysis. Your cDNA prep protocol might not be.
Is your cDNA...
Forum: Sample Prep / Library Generation 01-14-2019, 07:51 AM
Replies: 15
Views: 14,203
Posted By luc
Yep, the enzymatic fragmentation products...

Yep, the enzymatic fragmentation products generated by kits from from several vendors do not require any end-repair.
Forum: 454 Pyrosequencing 01-14-2019, 07:46 AM
Replies: 5
Views: 5,444
Posted By luc
In the age of long read sequencing (PacBio,...

In the age of long read sequencing (PacBio, Nanopore) and linked reads (10X Genomics), "mate pair" sequencing is as obsolete as 454.
Forum: Sample Prep / Library Generation 01-11-2019, 02:46 PM
Replies: 3
Views: 659
Posted By luc
Nextflex adapters are available for DNA...

Nextflex adapters are available for DNA applications and for RNA-seq applications. The latter are merely dilutions of the former, but Bioo provides the concentration info somewhere.
If you have...
Forum: Sample Prep / Library Generation 01-10-2019, 08:59 PM
Replies: 3
Views: 659
Posted By luc
Check the concentration of the Nextflex RNA-seq...

Check the concentration of the Nextflex RNA-seq adapters on their web page; I have not memorized it but it will work.
Forum: Pacific Biosciences 01-07-2019, 05:03 PM
Replies: 5
Views: 967
Posted By luc
Nothing about the read metrics raises warning...

Nothing about the read metrics raises warning flags - they could certainly have generated longer library molecules though since they did not multiplex the library.

Is the genome size about what...
Forum: Illumina/Solexa 12-29-2018, 12:39 PM
Replies: 9
Views: 1,667
Posted By luc
I would suggest to check the index reads for this...

I would suggest to check the index reads for this particular library to see which base is affected.
Forum: General 12-24-2018, 11:44 AM
Replies: 3
Views: 938
Posted By luc
Hi Kujin, to my knowledge the Lohman...

Hi Kujin,

to my knowledge the Lohman protocol should work, as is, on the iSeq - as any other Truseq library design. We do not have an iSeq however. Do you want to optimize it for paired-end...
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