SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Pacific Biosciences



Similar Threads
Thread Thread Starter Forum Replies Last Post
PacBio Library Prep workshop and PacBio SMART-Portal bootcamp - UC Davis - April 2015 DNATECH Events / Conferences 1 04-02-2015 09:33 AM
Bowtie output variability vcc Bioinformatics 0 01-11-2015 09:54 PM
How to create CCS from subreads without smrtcell data? bastianwur Pacific Biosciences 10 09-22-2014 02:37 AM
PacBio performance and efficiency in the field Trev Pacific Biosciences 2 04-23-2012 01:33 AM

Reply
 
Thread Tools
Old 10-13-2016, 08:53 AM   #1
ribodoc
Junior Member
 
Location: orange county, ca

Join Date: Dec 2011
Posts: 6
Default PacBio RS II SMRTcell performance variability

We are observing increased variability in the performance of SMRTcells by lot, strip and within a strip. Using the same SMRTbell template on different sequencing runs the variability can be as much as a two fold difference between strips of SMRTcells. We would like advice from other users who may experiencing the same issue.
ribodoc is offline   Reply With Quote
Old 10-13-2016, 11:59 AM   #2
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,358
Default

Are you referring to just successful loading? Or some other metrics (RL, accuracy)?
ECO is offline   Reply With Quote
Old 10-13-2016, 12:14 PM   #3
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,002
Default

@ribodoc: Was there a time when you were regularly getting consistent results and now you are not?
GenoMax is offline   Reply With Quote
Old 10-13-2016, 02:16 PM   #4
ribodoc
Junior Member
 
Location: orange county, ca

Join Date: Dec 2011
Posts: 6
Default

Quote:
Originally Posted by ECO View Post
Are you referring to just successful loading? Or some other metrics (RL, accuracy)?
Within a week, with the same SMRTbell template and loading concentration went from 47.44% P1 to 26.64% P1. Accuracy dipped from 0.841 to 0.831. Polymerase read length N50 had a smaller drop from 16285 to 15849.
ribodoc is offline   Reply With Quote
Old 10-13-2016, 02:40 PM   #5
ribodoc
Junior Member
 
Location: orange county, ca

Join Date: Dec 2011
Posts: 6
Default

Quote:
Originally Posted by GenoMax View Post
@ribodoc: Was there a time when you were regularly getting consistent results and now you are not?
Occasionally we see this occur and it was time to put it out to the community. We typically do a titration of SMRTbell concentration for loading and then do a production run on a strip of SMRTcells. There will be periods of time when the tritration and production runs are very consistent and then there are other times when they are not consistent and even not consistent within a strip of SMRTcells.
ribodoc is offline   Reply With Quote
Old 10-13-2016, 05:07 PM   #6
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,002
Default

Quote:
Originally Posted by ribodoc View Post
Occasionally we see this occur and it was time to put it out to the community. We typically do a titration of SMRTbell concentration for loading and then do a production run on a strip of SMRTcells. There will be periods of time when the tritration and production runs are very consistent and then there are other times when they are not consistent and even not consistent within a strip of SMRTcells.
That has been more or less the story. While I don't work at the bench I hear similar observations most of the time. There is no logical explanation that can be put forth either. Hopefully Sequel will be more reliable.
GenoMax is offline   Reply With Quote
Old 10-13-2016, 05:12 PM   #7
ribodoc
Junior Member
 
Location: orange county, ca

Join Date: Dec 2011
Posts: 6
Default

Quote:
Originally Posted by GenoMax View Post
That has been more or less the story. While I don't work at the bench I hear similar observations most of the time. There is no logical explanation that can be put forth either. Hopefully Sequel will be more reliable.
Thank you for the response.
ribodoc is offline   Reply With Quote
Old 10-14-2016, 10:10 AM   #8
ECO
--Site Admin--
 
Location: SF Bay Area, CA, USA

Join Date: Oct 2007
Posts: 1,358
Default

@Genomax...the logical explanation is that the SMRTcell production process is dramatically more complicated than all other sequencing consumables, ZMW size and uniformity, the micromirror shape, aluminum coating, then on top of that the blocking of the surface and then the biotinylation.

I know it's no solace to scientists trying to do reproducible production (we run two RSII's all the time), but it's kind of amazing just how well it all works, and is a testament to all the people that have contributed at PACB. (disclaimer I worked there for a long time and know a lot of people there still)
ECO is offline   Reply With Quote
Old 10-14-2016, 11:05 AM   #9
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 7,002
Default

I have nothing but admiration for the amount of technology that went into making the original PacBio machine, consumables.

Based on anecdotal references keeping an RSII running ALL the time seems to be essential for its well being. Sadly we could not do that.
GenoMax is offline   Reply With Quote
Old 10-14-2016, 02:13 PM   #10
luc
Senior Member
 
Location: US

Join Date: Dec 2010
Posts: 422
Default

The SMRT-cell quality (and correlated to it the yields) can indeed be quite variable in general .
The very last batches of SMRT-cells seem to be more variable than usual (for us and also some other labs). These higher frequency problems seem to occur at least once a year.
luc 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 04:26 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO