SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
Titanium Amplicon run. gilly 454 Pyrosequencing 42 03-28-2012 09:21 AM
Are Illumina library fragment lengths actually normally distributed? delphi_ote Sample Prep / Library Generation 6 05-09-2011 01:01 PM
454 Roche Titanium Run Costs gendxdoc 454 Pyrosequencing 0 05-03-2010 03:41 PM
GS FLX Titanium series and small fragment doc_lille 454 Pyrosequencing 0 03-22-2010 09:23 AM
454 runAssembly contig lengths DNAjunk 454 Pyrosequencing 8 08-30-2009 10:55 PM

Reply
 
Thread Tools
Old 05-16-2011, 04:15 PM   #1
Tom McFarland
Junior Member
 
Location: Hawaii

Join Date: May 2011
Posts: 2
Default Multiple fragment lengths in single 454 titanium run?

Hi all,

I have two different fragments that I want to sequence with 454 titanium. One is about 400 bp long and the other about 500 bp long (including gene-specific primers but not fusion primers). Can I run these together in a single run? A lab mate told me that a disproportionately high number of reads will come from the shorter fragment, but it seems a lot of people are doing multiple fragment lengths and getting good results. I have about 100 samples of the 400 bp fragment and about 60 samples of the 500 bp fragment and was hoping to get by with a 1/4 plate run.

Thanks for any help.
Tom McFarland is offline   Reply With Quote
Old 05-17-2011, 11:04 AM   #2
Nicole 454 Sequencing
Member
 
Location: Branford

Join Date: Apr 2011
Posts: 19
Default

Hi Tom,

We recommend that amplicons be designed between 200-600 base pairs and within approximately 150 base pairs of each other. Smaller fragments will be preferentially amplified when fragment lengths span outside of these recommendations. With that said, your fragments of 400 bp and 500 bp can be sequenced in a single run.

APP No. 001-2010 gives some guidance on Amplicon Sequencing using the GS FLX Titanium platform. This Application Brief and additional references can be found on our customer-accessible website at www.454.com/my454.

Please don’t hesitate to contact your local Roche representative with further queries.

Kind Regards,

Nicole
Technical Support Scientist
454 Life Sciences, A Roche Company
Nicole 454 Sequencing is offline   Reply With Quote
Old 05-17-2011, 08:57 PM   #3
Tom McFarland
Junior Member
 
Location: Hawaii

Join Date: May 2011
Posts: 2
Default Thanks

Thank you for your help.
Tom McFarland is offline   Reply With Quote
Old 05-18-2011, 06:47 AM   #4
vlee2
Member
 
Location: Richmond, VA

Join Date: Apr 2009
Posts: 19
Default

When we sequence different sizes of amplicons, we separate them in groups by sizes, emPCR, and then combine DNA beads together for sequencing.
vlee2 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 01:04 PM.


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