SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Oxford Nanopore



Similar Threads
Thread Thread Starter Forum Replies Last Post
Pacbio sequel for single cell human WGS? WhatsOEver Pacific Biosciences 5 06-27-2017 12:02 PM

Reply
 
Thread Tools
Old 05-12-2017, 07:20 AM   #41
Ola
Member
 
Location: Sweden

Join Date: Aug 2011
Posts: 30
Default

Quote:
Originally Posted by WhatsOEver View Post
How does this fit together?
Could you tell us where you got this information from?
I would especially be interested in seeing some real data from this machine.
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 much of it at this point, they showed a bit about how it works and that it can generate data. I don't think they aim for WGS, more like gene panels and specific clinical tests.
Ola is offline   Reply With Quote
Old 05-12-2017, 07:29 AM   #42
gringer
David Eccles (gringer)
 
Location: Wellington, New Zealand

Join Date: May 2011
Posts: 799
Default

Quote:
Originally Posted by ymc View Post
I am interested in working on samples with around 10 human cells that share the same DNA (barring mosaicism). It will be great if you also give this scenario a try and tell us if we can get any sequences from the ten cells.
Amplification will be needed from 10 cells as well. The lower limit currently for nanopore (allowing for a bit of a drop in yield) is about 10,000 cells.

The rapid sequencing kit needs about 200ng of high-quality DNA for sequencing reads of about 1-10kb, but it's possible to go down to about 10% of that before the drop in run yield falls off a cliff.
gringer is offline   Reply With Quote
Old 05-12-2017, 10:42 AM   #43
Brian Bushnell
Super Moderator
 
Location: Walnut Creek, CA

Join Date: Jan 2014
Posts: 2,695
Default

Quote:
Originally Posted by WhatsOEver View Post
You cannot map short reads containing only repetitive sequence to a long read spanning the repetitive region, can you? If you could, you would be able to map the same short read to the reference assembly - which you can't. Or am I missing something important in your statement?
I know it sounds odd, but yes, you can. Imagine two long reads covering different repeats (or different ploidies). Short reads cannot map to the assembly and differentiate which one they should map to, because in the assembly, they look the same. But if there is an actual SNP in one copy, then reads with that SNP will (in general) map to the copy with the SNP, and reads without the SNP will (in general) map to the copy without the SNP. It's not perfectly clean due to the noisiness of the long reads, but with sufficient copies, on average, the version with the SNP will get corrected to have the SNP, and the version without the SNP will get corrected to the reference. Then, aligning the corrected long reads will allow you to properly place the SNP in the correct copy. I've done this with Poplar PacBio + Illumina libraries for the purpose of phasing.
Brian Bushnell is offline   Reply With Quote
Old 05-19-2017, 09:02 PM   #44
gringer
David Eccles (gringer)
 
Location: Wellington, New Zealand

Join Date: May 2011
Posts: 799
Default

Here's one 222kb read [6a6944fa-9143-490f-b5dd-22e7e7267d3c_Basecall_1D_template] from the nanopore-wgs sequencing project, run FAF09968. The resolution of this is 109 bases per pixel, so the repeat unit length is around 2500bp:

http://i.imgur.com/5MNzBsf.png

Smaller version:



Unfortunately, I haven't yet been able to find a completely covered repeat, but hopefully this is sufficient to demonstrate the sequencing issue with other platforms.

For additional reference, here is the WGS consortium github page:

https://github.com/nanopore-wgs-consortium/NA12878

Direct link to this run fastq file:

http://s3.amazonaws.com/nanopore-hum...09968.fastq.gz

The read itself can be found here:

http://www.gringene.org/data/largeRepeat_6a69.fa.gz

Last edited by gringer; 05-19-2017 at 09:08 PM.
gringer is offline   Reply With Quote
Old 05-19-2017, 09:26 PM   #45
gringer
David Eccles (gringer)
 
Location: Wellington, New Zealand

Join Date: May 2011
Posts: 799
Default

Quote:
Originally Posted by gringer View Post
Unfortunately, I haven't yet been able to find a completely covered repeat
... and I just found one (308kb read). The main repeat region has unique sequence outside it (e.g. where that line of blue is):

http://i.imgur.com/aym9zTg.png



Read is here:

http://www.gringene.org/data/largeRepeat_6cec.fa.gz
gringer is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 02:50 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO