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Forum: Illumina/Solexa 01-16-2018, 04:55 PM
Replies: 29
Views: 4,393
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, 12:55 PM
Replies: 13
Views: 1,008
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, 05:31 PM
Replies: 0
Views: 181
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, 05:17 PM
Replies: 3
Views: 219
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, 09:44 AM
Replies: 13
Views: 1,008
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, 09:26 AM
Replies: 13
Views: 1,008
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, 08:19 PM
Replies: 3
Views: 219
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, 10:06 AM
Replies: 20
Views: 10,668
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, 02:27 PM
Replies: 3
Views: 648
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, 05:42 PM
Replies: 4
Views: 1,052
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, 06:44 PM
Replies: 4
Views: 606
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, 04:35 PM
Replies: 1
Views: 519
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, 04:29 PM
Replies: 4
Views: 590
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, 01:42 AM
Replies: 4
Views: 774
Posted By luc
Thanks Joseph! Have you perhaps tried...

Thanks Joseph!
Have you perhaps tried extending the UMI a bit?
Forum: Sample Prep / Library Generation 11-03-2017, 01:36 AM
Replies: 2
Views: 458
Posted By luc
The sequences of Kapa, TrueSeq and...

The sequences of Kapa, TrueSeq and BiooScientific adapters are identical, except perhaps some index sequences. They are compatible, you might have to figure out the appropriate concentrations.
Forum: RNA Sequencing 11-02-2017, 06:00 AM
Replies: 4
Views: 774
Posted By luc
Thanks for the information Joseph, I would...

Thanks for the information Joseph,

I would have two questions:
Why should the Smart oligos be 5' biotinylated?
How much of a balanced library do you need to spike in to sequence through the...
Forum: Pacific Biosciences 11-01-2017, 12:50 AM
Replies: 4
Views: 608
Posted By luc
10 to13 kb libraries sounds a bit short? Which...

10 to13 kb libraries sounds a bit short? Which length were the samples sheared for and which cut did you use for the pippin sise-selection?
Forum: Illumina/Solexa 10-23-2017, 11:11 PM
Replies: 15
Views: 5,105
Posted By luc
The HiSeq 4000 works now quite well with low...

The HiSeq 4000 works now quite well with low complexity libraries. There was a software update about a year ago.
Forum: Illumina/Solexa 10-11-2017, 04:57 PM
Replies: 26
Views: 2,806
Posted By luc
I am not sure that index hopping onto PhiX reads...

I am not sure that index hopping onto PhiX reads would be a good measure. As far as I remember the Illumina PhiX adapter sequences are quite different from TruSeq or Nextera sequences.
Forum: RNA Sequencing 10-10-2017, 07:06 PM
Replies: 2
Views: 745
Posted By luc
Hi Jin1, I assume you will be pooling...

Hi Jin1,

I assume you will be pooling multiple libraries? ; thus, the molarity of the final pool will have to meet the requirements of your sequencing service -- not the individual libraries.
If...
Forum: General 10-06-2017, 05:18 PM
Replies: 3
Views: 652
Posted By luc
If you count like Illumina does, then yes these...

If you count like Illumina does, then yes these would be 800M reads. You will still sequence only 400M library molecules though.
Forum: Illumina/Solexa 10-03-2017, 06:30 PM
Replies: 3
Views: 779
Posted By luc
Indeed very weird. Fortunately we have not seen...

Indeed very weird. Fortunately we have not seen this problem so far.
Are you using custom sequencing primers?
Forum: Sample Prep / Library Generation 10-03-2017, 06:01 PM
Replies: 1
Views: 415
Posted By luc
The bonds between these bases are...

The bonds between these bases are phosphorothioated to prevent nuclease digestion (e.g. from proofreading polymerases) : https://www.idtdna.com/site/Catalog/Modifications/Category/8
Forum: Sample Prep / Library Generation 09-30-2017, 03:48 PM
Replies: 6
Views: 703
Posted By luc
Yes. That is, what I meant. 10X Genomics uses a...

Yes. That is, what I meant. 10X Genomics uses a 10 nt long UMI for their single-cell kit.
Forum: Sample Prep / Library Generation 09-29-2017, 10:26 AM
Replies: 6
Views: 703
Posted By luc
You could incorporate a stretch of "N"s - lets...

You could incorporate a stretch of "N"s - lets say eighth of them as UMI. This would be by far the cheapest option.
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