SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
Illumina small RNA ladder and NEB miRNA marker lostviking Sample Prep / Library Generation 2 08-18-2014 10:41 AM
NEB multiplex adapter kit murpheli Illumina/Solexa 9 10-21-2013 09:29 AM
NEBNext ChIP-Seq library prep kit vs Illumina TruSeq kit? ASL Sample Prep / Library Generation 1 04-10-2013 07:35 PM
KAPA qPCR kit for XL+ libraries? madseq 454 Pyrosequencing 2 08-31-2012 07:39 AM
NEB lib prep kit BIG_SNP Illumina/Solexa 0 08-20-2009 02:34 PM

Reply
 
Thread Tools
Old 09-27-2013, 12:54 PM   #1
mkalive73
Junior Member
 
Location: Arizona

Join Date: Sep 2013
Posts: 1
Default using NEB kit to make libraries for Illumina MiSeq

Hello,

I used NEB multiplex kit to make libraries for Illumina MiSeq. Trying to figure out the amount of sample to load in the flow cell to get a decent cluster density. Last round I loaded 12pM of total sample and it over clustered with my coverage very low. My colleague uses 7pM of the same kind of sample on a HiSeq, how does that compare with MiSeq? should Miseq sample amount be lesser or higher?

Thanks for your suggestions.
mkalive73 is offline   Reply With Quote
Old 09-30-2013, 07:54 AM   #2
Isequencestuff
Member
 
Location: Cambridge

Join Date: Nov 2012
Posts: 21
Default

what MiSeq kit (flowcell) are you using? I assume you're using the V2 kit since you're aiming to load 12 pM of total sample. If your quantitation method is accurate, aiming to load 12 pM should give you a perfect cluster density. We usually get around 850-900. Our quantification process: qPCR post library construction, then based on those quantities dilute all library samples to 2 nM and pool. Then run a last qPCR on that one pooled library sample to make sure it's close to 2 nM (this saves us from wasting a flow cell). From there we dilute to 12 pM final (including about 1-5% phiX), then load and go.
Isequencestuff is offline   Reply With Quote
Old 09-30-2013, 09:58 PM   #3
MWN
Junior Member
 
Location: CA

Join Date: Aug 2011
Posts: 8
Default

For V2, I load 11 pM based on Qubit. For V3, 14 pM.
MWN is offline   Reply With Quote
Old 10-01-2013, 10:13 AM   #4
kcchan
Senior Member
 
Location: USA

Join Date: Jul 2012
Posts: 184
Default

Comparing loading concentrations isn't very beneficial. Long inserts also don't behave in the same way as short inserts, so a 12pM TruSeq DNA library may be fine whereas a 12pM miRNA library will overcluster. Also, each instrument has its own different sweet spot. The same sample at 12pM may be perfect on one MiSeq but overcluster on a different MiSeq. It's better being more conservative (4-6pM) and working your way up over the course of a few runs.
kcchan 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 01:02 PM.


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