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Forum: Introductions 10-15-2017, 11:47 AM
Replies: 3
Views: 435
Posted By Ola
Hi, adding a larger volume would make it...

Hi,

adding a larger volume would make it easier to amplify even though the concentration is the same since you then will have more template molecules. It is also possible to amplify from less than...
Forum: General 10-04-2017, 11:28 PM
Replies: 14
Views: 1,594
Posted By Ola
That is what I am using (with 2x16G), excellent...

That is what I am using (with 2x16G), excellent performance for the price.
Forum: Sample Prep / Library Generation 07-06-2017, 03:12 PM
Replies: 3
Views: 849
Posted By Ola
Thanks for the link. As said above, make sure...

Thanks for the link. As said above, make sure your buffer is 16% PEG and 0.6M NaCl if you are using it as 1x.
Forum: Sample Prep / Library Generation 05-25-2017, 01:20 PM
Replies: 10
Views: 1,581
Posted By Ola
Ethanol concentration in washes is not a problem...

Ethanol concentration in washes is not a problem with HMW DNA. Long elutions at 37* may help, just like for dissolving a HMW pellet after ethanol precipitation. Sometimes it seems you really need to...
Forum: Oxford Nanopore 05-12-2017, 07:20 AM
Replies: 44
Views: 3,801
Posted By Ola
If you make 10000 measurements per second and...

If you make 10000 measurements per second and read DNA at a slow speed it will generat lots of raw data but not so much sequence. They presented early data at a VIB conference. I would not make too...
Forum: Oxford Nanopore 05-10-2017, 12:58 PM
Replies: 44
Views: 3,801
Posted By Ola
From what I have seen they can generate a ton of...

From what I have seen they can generate a ton of raw data and some sequence (bacterial genomes), but the quality per read is not impressive (yet). It relies on a polymerase which is much slower than...
Forum: Oxford Nanopore 05-09-2017, 03:02 AM
Replies: 44
Views: 3,801
Posted By Ola
WhatsOEver, you are correct in that PacBio at...

WhatsOEver, you are correct in that PacBio at this point gives a much cleaner alignment but of course with amplified DNA you will get lower throghput given the limited read numbers. For ONT...
Forum: Oxford Nanopore 04-25-2017, 07:20 AM
Replies: 1
Views: 769
Posted By Ola
Hi! You can ligate the BCA to any DNA and then do...

Hi! You can ligate the BCA to any DNA and then do a barcoded PCR, e.g if you want to use genomic DNA or have amplicons without the ONT pcr-priming tail. We also tend to get lower than recommended...
Forum: Bioinformatics 04-10-2017, 03:45 AM
Replies: 9
Views: 786
Posted By Ola
Discovar de novo was designed specifically for...

Discovar de novo was designed specifically for 2x250 bp reads so you could give it a try. Of course it is recommended to have inserts longer than the sequencing reads (500 bp in your case) but it...
Forum: Oxford Nanopore 02-05-2017, 11:08 AM
Replies: 9
Views: 2,145
Posted By Ola
I checked error rates (bwa mem -x ont2d + picard...

I checked error rates (bwa mem -x ont2d + picard CollectAlignmentSummary) for an amplicon run now, with 145 2D reads for a 8 kb human region. It gave 1.5% subst error and 3.2 % indel errors. This was...
Forum: Oxford Nanopore 02-03-2017, 03:39 AM
Replies: 9
Views: 2,145
Posted By Ola
Good question. For 1D it is ~10-12% now, somewhat...

Good question. For 1D it is ~10-12% now, somewhat sample/sequence and alignment dependent. That would give you ~5-fold more data compared to 2D runs, but if you plan for barcoding and don't need the...
Forum: Oxford Nanopore 01-05-2017, 11:24 AM
Replies: 11
Views: 2,606
Posted By Ola
Hi! 1. This is the sequencing speed. 2D runs at...

Hi!
1. This is the sequencing speed. 2D runs at 250 bases/s while 1D now goes through at 450 bps.
2. You would have to post on the forum and ask what others think... It would be great if Metrichor...
Forum: Oxford Nanopore 10-27-2016, 01:58 AM
Replies: 4
Views: 1,422
Posted By Ola
Sure, you can load whatever you have. 60 ng...

Sure, you can load whatever you have. 60 ng should be fine. In case you get poor yields (i.e have many single pores but few in strand) you can just stop the run and add a new library. I have not...
Forum: Oxford Nanopore 10-09-2016, 12:52 PM
Replies: 3
Views: 2,320
Posted By Ola
10 Gb would be for 1d runs at 450 bps and is...

10 Gb would be for 1d runs at 450 bps and is likely the total basecalls they achieved in their best runs. It does look like significant improvements in yield and and stability of the flowcells and...
Forum: Bioinformatics 06-07-2016, 04:02 AM
Replies: 1
Views: 620
Posted By Ola
Yes, that would be a mate-pair library. Note...

Yes, that would be a mate-pair library.

Note that Broad moved from Allopaths to Discover with 2x250 bp reads for the 200 mammals project, so may be worth looking into if that fits in your budget....
Forum: Sample Prep / Library Generation 05-25-2016, 01:13 PM
Replies: 5
Views: 1,016
Posted By Ola
The first sample has a very high adaptor dimer...

The first sample has a very high adaptor dimer peak. Qubit only measures total DNA.
Forum: Oxford Nanopore 05-18-2016, 03:09 AM
Replies: 7
Views: 2,435
Posted By Ola
Just to be clear, the MinION early access program...

Just to be clear, the MinION early access program ended last summer. You can now register for access to the community and get a mk1 + start kit (2 flow cells + reagents) for $1000. Individual...
Forum: Oxford Nanopore 05-18-2016, 12:00 AM
Replies: 4
Views: 1,326
Posted By Ola
The 2D read is basecalled from the signal from...

The 2D read is basecalled from the signal from both strands so it is from the same molecule. Reads with failed 2D basecalls should be discarded as the hairpin may not have been correctly identified....
Forum: Oxford Nanopore 05-17-2016, 02:31 AM
Replies: 4
Views: 1,326
Posted By Ola
They should not with the current chemistry since...

They should not with the current chemistry since the motor protein sits on the adapter for the template strand.
Forum: Oxford Nanopore 05-17-2016, 02:25 AM
Replies: 7
Views: 2,435
Posted By Ola
The big leap is with the new pore (R9) and base...

The big leap is with the new pore (R9) and base callers that are being released now. This chemistry also runs faster, at 250 b/s vs previous 70 b/s. Yield between flowcells is still an issue but...
Forum: Pacific Biosciences 03-23-2016, 11:34 AM
Replies: 50
Views: 8,694
Posted By Ola
No, that is an old post describing the...

No, that is an old post describing the introduction of the RS system. Things have changed a lot: "Read lengths decay exponentially -- but with lots around 1Kb and quite a few around 5K", "The...
Forum: Pacific Biosciences 03-22-2016, 06:19 AM
Replies: 50
Views: 8,694
Posted By Ola
One limitation with the PacBio is that it relies...

One limitation with the PacBio is that it relies on very long reads since it (currently) only does one read per ZMW, and increased read lengths does not increase throughput if you can't make...
Forum: Pacific Biosciences 03-21-2016, 11:47 AM
Replies: 50
Views: 8,694
Posted By Ola
That is good to know, I was not aware of the...

That is good to know, I was not aware of the differences in optics between the two systems. It is great if it really is as scalable as you say.

It may well be difficult to make reliable...
Forum: Pacific Biosciences 03-20-2016, 01:34 PM
Replies: 50
Views: 8,694
Posted By Ola
I guess it is because they have their own...

I guess it is because they have their own community so you are more likely to get a response there. If you have not already, now would be a good time to try out the MinION. I am sure you could spare...
Forum: Sample Prep / Library Generation 01-11-2016, 04:27 AM
Replies: 5
Views: 1,062
Posted By Ola
I have used this with good results: ...

I have used this with good results:

http://www.thermofisher.com/order/catalog/product/61012?ICID=search-product
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