SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Ion Torrent



Similar Threads
Thread Thread Starter Forum Replies Last Post
Difference between Ion torrent proton and Ion PGM Bionerd Ion Torrent 7 09-30-2015 02:55 PM
ion proton and torrent variant caller Rabu Bioinformatics 0 11-19-2014 07:02 AM
Ion Torrent libraries on proton? Apex Ion Torrent 2 12-12-2012 07:33 AM
Ion Torrent $1000 Genome!? Benchtop Ion Proton Sequencer aeonsim Ion Torrent 88 10-28-2012 05:50 AM

Reply
 
Thread Tools
Old 12-15-2015, 03:38 AM   #1
zcm2403
Junior Member
 
Location: china

Join Date: Dec 2014
Posts: 9
Default ion torrent proton QC problem

Hi,everyone!
I had ran a p1 chip yesterday.After running completed i found that the on target reads and uniformity was very low,just as shown in the picture.Another problem is that my designed amplicon length is about 300 bp,however the results reads mean length is only 150 bp.There is something wrong in my experiments.could someone give me some suggestions?
THANKS!
Attached Images
File Type: jpg IMG_20151215_154007-1.jpg (97.6 KB, 40 views)
File Type: jpg IMG_20151215_154130-4.jpg (56.1 KB, 28 views)
zcm2403 is offline   Reply With Quote
Old 12-17-2015, 04:53 AM   #2
Xray1
Junior Member
 
Location: Germany

Join Date: Jun 2015
Posts: 5
Default

I have no Proton experience myself, but max read length of Proton is 200bp, isn't it?
What type of application is this, Ampliseq or something else?
Xray1 is offline   Reply With Quote
Old 12-17-2015, 06:28 AM   #3
zcm2403
Junior Member
 
Location: china

Join Date: Dec 2014
Posts: 9
Default

thanks for your apply.My experiment is Ampliseq.And just as shown in the picture,the max length is about 350bp.I think the cause is that the long reads is too less.
zcm2403 is offline   Reply With Quote
Old 12-17-2015, 10:08 AM   #4
Xray1
Junior Member
 
Location: Germany

Join Date: Jun 2015
Posts: 5
Default

What panel? As far as I can tell, Ampliseq research panels (those designed by Ion or community panels) are usually 125-275bp amplicon range. These should work on Proton.

Since Pipeline Version 4, the Ampliseq designer allows you to use long amplicons with 125-375bp amplicons. These panels will only work on PGM or S5-520/530 with HiQ-Chemistry. These panels are nice, as you usually get better design coverage. But they have these limitations (also not usable with equalizer kit and therefore probably not usable with IonChef Ampliseq library prep). Ion/Thermo likes to forget about these limitations in their documentation, like no word on this in the Ampliseq Kit documentation. But in the Ampliseq designer, it will tell you what machine/chip you can use the panel.
Xray1 is offline   Reply With Quote
Old 12-17-2015, 06:12 PM   #5
zcm2403
Junior Member
 
Location: china

Join Date: Dec 2014
Posts: 9
Default

THANKS!I had used the HiQ kit with 375bp amplicons on the proton before.The results was good.I do not understand why the on target reads and the uniformity was so bad in this panel?I mixed some short length pannel with long length panel.Is it the keypoint?
zcm2403 is offline   Reply With Quote
Old 12-18-2015, 03:34 AM   #6
Xray1
Junior Member
 
Location: Germany

Join Date: Jun 2015
Posts: 5
Default

When I get uniformity near 50%, it is usually just that one of two primer pools didn't work. You can check whether amplicons with low coverage are all from the same pool. If so, your ampliseq library prep just didn't work.

Mixing libraries generated with long and short amplicon panels in the templating step might add to the problem. However, most "long amplicon" panels also have shorter amplicons and still work just great on the PGM. I am currently running a long amplicon panel with the shortest amplicon somewhere around 130pb and it shows great results. However, for Chef/PGM (and I think also the OT2) templating settings are different for 200bp and 400bp runs. For Proton you probably always use the same templating and that might cause more proplems when you add more short amplicons. Make sure a certain long amplicon panel works fine on the Proton before mixing with other panels.

You will have to try around a bit I guess, since the long panels are not officially supported. Interesting to read that it can work. Getting longer reads on Proton was one of the things people really hoped for (besides new P-chips.....), so the fact that Ion never got anything going (not even on the S5-540) indicated to me that it just didn't work. I guess most people therefore just avoid even trying this......Do you adjust any of your protocols (amount of library you put in or whatever) to get the long amplicons running?
Xray1 is offline   Reply With Quote
Old 12-18-2015, 05:06 AM   #7
zcm2403
Junior Member
 
Location: china

Join Date: Dec 2014
Posts: 9
Default

Thanks for your suggestion.Before running on the proton i did a preliminary experiments on the PGM.And the uniformity was about 85%.So the results was good.Longer reads panel works well on the proton in our lab.And except the HiQ kit we did all the experiments following the protocals.
zcm2403 is offline   Reply With Quote
Old 02-09-2017, 08:15 AM   #8
Isabella97
Junior Member
 
Location: Cracow

Join Date: Feb 2017
Posts: 1
Default

Thanks. I was looking for this answer for a long time.
Isabella97 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 06:04 PM.


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