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Forum: RNA Sequencing 07-13-2018, 05:53 PM
Replies: 1
Views: 401
Posted By luc
Prolonged high heat exposure definitely...

Prolonged high heat exposure definitely introduces a lot of damages to DNA. I would expect it to be pretty fragmented by now. I also assume the Nextera transpose would require dsDNA - thus, you...
Forum: Sample Prep / Library Generation 06-16-2018, 06:08 PM
Replies: 3
Views: 496
Posted By luc
Give it more time and vortex once in a while. ...

Give it more time and vortex once in a while. They will resuspend in the end.
Forum: Bioinformatics 06-15-2018, 05:16 PM
Replies: 1
Views: 341
Posted By luc
An DNA oligo with a rGrGrG 3'-end ((the template...

An DNA oligo with a rGrGrG 3'-end ((the template switching oligo) will anneal to the CCC overhang and the RT will continue to work on this newly added template. This allows you to add a defined...
Forum: Ion Torrent 06-15-2018, 05:09 PM
Replies: 3
Views: 644
Posted By luc
A neighboring lab has the OT-One. This first...

A neighboring lab has the OT-One. This first version is unusable for just about anything - it can't hold the pipettes properly and the plates have a lot of play. Version two looks very different,...
Forum: Illumina/Solexa 05-31-2018, 05:08 PM
Replies: 1
Views: 297
Posted By luc
Is this a one-off experiment or are you planning...

Is this a one-off experiment or are you planning for multiple samples?

You will need to shear the DNA sample either by sonication or enzymatically to pieces of about 300 to 450 bp length for the...
Forum: Bioinformatics 04-22-2018, 02:16 PM
Replies: 13
Views: 1,456
Posted By luc
Final costs depend on reagent costs and yields...

Final costs depend on reagent costs and yields (and reliability). Results for both systems will depend a lot on DNA sample quality. At current yields the Sequel runs will yield more data/dollar in...
Forum: Bioinformatics 04-20-2018, 04:16 PM
Replies: 13
Views: 1,456
Posted By luc
The sequencer is certainly cheap, but to my...

The sequencer is certainly cheap, but to my knowledge not the reagents and additional flowcells. PacBio might be more affordable?
Forum: Illumina/Solexa 04-13-2018, 06:01 PM
Replies: 2
Views: 672
Posted By luc
It could work - however you will not be able to...

It could work - however you will not be able to sue the PhiX libraries as spike in since you will can't use the Illumina sequencing primers together with your extended versions.
Forum: Bioinformatics 04-06-2018, 03:23 PM
Replies: 636
Views: 125,960
Posted By luc
Convenient way to avoid re-indexing references? ...

Convenient way to avoid re-indexing references?

When working with big genomes one would really like to avoid re-indexing reference sequences. BBmap is always looking for the reference indices in...
Forum: Sample Prep / Library Generation 03-31-2018, 10:22 AM
Replies: 5
Views: 1,364
Posted By luc
It is unlikely that the Tn5 will cut/tagment your...

It is unlikely that the Tn5 will cut/tagment your 113 bp fragment twice - this would be required for sequencing.
Forum: Sample Prep / Library Generation 03-26-2018, 05:58 PM
Replies: 1
Views: 724
Posted By luc
The NEBNext® End Repair Module is missing the...

The NEBNext® End Repair Module is missing the A-tailing component (likely taq ploymerase).
Forum: Illumina/Solexa 01-16-2018, 03:55 PM
Replies: 29
Views: 5,761
Posted By luc
Hi ngagnes, in our experience the reagent...

Hi ngagnes,

in our experience the reagent quality has gotten better, but is still not as good as 3 years ago and we still see our low quality run outliers (that should have performed better...
Forum: Illumina/Solexa 01-10-2018, 11:55 AM
Replies: 13
Views: 1,983
Posted By luc
I guess the libraries are the same as for the...

I guess the libraries are the same as for the other Illumina sequencers?
Forum: Sample Prep / Library Generation 01-09-2018, 04:31 PM
Replies: 0
Views: 546
Posted By luc
AmpliSeq - what makes it special?

Hi All,

I might be a bit dense. Could you help me understand what makes the AmpliSeq protocol special?


It seems to use multiplexed PCR primers with universal tags.
These universal tags...
Forum: Illumina/Solexa 01-09-2018, 04:17 PM
Replies: 3
Views: 528
Posted By luc
Hi liaoyunshi, sorry, I do not understand...

Hi liaoyunshi,

sorry, I do not understand what you are trying to achieve with the inline barcodes.
Working with the tru-seq style barcodes is preferable for all applications I can think off.
...
Forum: Illumina/Solexa 01-09-2018, 08:44 AM
Replies: 13
Views: 1,983
Posted By luc
This has more details: ...

This has more details:
http://omicsomics.blogspot.com/2018/01/iseq.html#more

If the flowcell price is correct it seems that the instrument is currently only of interest in cases where speed is of...
Forum: Illumina/Solexa 01-09-2018, 08:26 AM
Replies: 13
Views: 1,983
Posted By luc
I guess the low instrument cost and the small...

I guess the low instrument cost and the small size are supposed to sway people who are impressed by the nanopore instruments? despite the data being of completely different nature.
I twill totally...
Forum: Illumina/Solexa 01-08-2018, 07:19 PM
Replies: 3
Views: 528
Posted By luc
Hi liaoyunshi, Adding inline barcodes by...

Hi liaoyunshi,
Adding inline barcodes by ligation requires more effort (synthesis of two partially complementary oligos for each barcode) than adding truseq-style barcodes. Is there a special...
Forum: Illumina/Solexa 12-29-2017, 09:06 AM
Replies: 20
Views: 12,971
Posted By luc
As mentioned above, the two peaks could very well...

As mentioned above, the two peaks could very well be a sign of a mixed sample (contamination).
You could remove the all the high GC content reads and see if this improves the assembly.
BBtools...
Forum: Bioinformatics 12-26-2017, 01:27 PM
Replies: 3
Views: 1,334
Posted By luc
Anybody looking at this would need some more...

Anybody looking at this would need some more info: What type of organism? Which genome size is expected? What read lengths did you use? What library prep protocol and insert sizes?
Forum: Sample Prep / Library Generation 12-22-2017, 04:42 PM
Replies: 4
Views: 1,647
Posted By luc
I have not done any tests. I guess the assumption...

I have not done any tests. I guess the assumption is that the oligos will make multiple attempts to hybridize and that a two-base anchor is "stronger" than a single base.
Forum: Sample Prep / Library Generation 11-22-2017, 05:44 PM
Replies: 4
Views: 958
Posted By luc
As consequence from what Eric wrote, one often...

As consequence from what Eric wrote, one often has to eyeball the RNA integrity for insect samples on the Bioanalyzer (I am looking ratio of the rRNA peak size to the smaller debris fragments).
Your...
Forum: Sample Prep / Library Generation 11-22-2017, 03:35 PM
Replies: 5
Views: 2,018
Posted By luc
Yep, we have been using MagBio's HighPrep PCR...

Yep, we have been using MagBio's HighPrep PCR beads and the KapaPure beads. Both worked similar to the Ampure XP beads, also for size selections.
Forum: Pacific Biosciences 11-22-2017, 03:29 PM
Replies: 4
Views: 1,220
Posted By luc
Markiyan has alluded to it already; Pacbio data...

Markiyan has alluded to it already; Pacbio data are not suitable for genome size estimates based on kmer analyses. The error rates of the uncorrected raw data are too high.
Forum: RNA Sequencing 11-03-2017, 12:42 AM
Replies: 4
Views: 1,438
Posted By luc
Thanks Joseph! Have you perhaps tried...

Thanks Joseph!
Have you perhaps tried extending the UMI a bit?
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