SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
Stopped MiSeq run memento Illumina/Solexa 24 10-28-2015 07:17 PM
MiSeq stopped run yaximik Illumina/Solexa 20 03-18-2013 07:10 AM
How much PhiX in MiSeq run? thdybwf Illumina/Solexa 1 11-26-2012 03:01 AM
Maximum number of cycles from 300-cycle MiSeq kit... ECO Illumina/Solexa 13 11-09-2012 04:51 AM
Worst MiSeq Run ever? pmiguel Illumina/Solexa 0 04-30-2012 04:54 AM

Reply
 
Thread Tools
Old 04-14-2013, 10:59 AM   #1
Nikvailo
Member
 
Location: -

Join Date: Apr 2013
Posts: 15
Post Maximum Sample per MiSeq Run

Hi,

In both of MiSeq manual and Custom Amplicon Prep. Guide, it is said that maximum 96 samples can be read per run. It is seen that number of i5 and i7 primers (w/ indices) limits the number of samples.

There are 8 different i5 primers and 12 different i7 primers and combination of these create tags for 96 samples. Is it possible to design additional i5 or i7 primers (w/ custom indices) to increase sample number that can be read per run?

Thank you,

Last edited by Nikvailo; 04-14-2013 at 11:14 AM.
Nikvailo is offline   Reply With Quote
Old 04-22-2013, 11:29 AM   #2
IDDan
Junior Member
 
Location: Gem State

Join Date: Apr 2013
Posts: 5
Default

We do MiSeq runs with 300+ amplicons per run. We are currently limited by primer cost, not the HiSeq hardware/software. The keys are (IMO):
1) having the bioinformatic support to sort out the reads when the run is done
2) properly designing barcodes/indexes/tags so that they are sufficiently difference from each other to believe custom sample barcodes (97+) wouldn't be mis-called as barcode 1-96 or (each other) when you are sorting out which reads go to which barcodes.
IDDan is offline   Reply With Quote
Old 04-22-2013, 12:44 PM   #3
JackieBadger
Senior Member
 
Location: Halifax, Nova Scotia

Join Date: Mar 2009
Posts: 381
Default

It all depends on the depth you want to achieve per amplicon.
We run ~ 1000 samples per run achieving depths in the 1000s
JackieBadger is offline   Reply With Quote
Old 04-23-2013, 06:26 AM   #4
thomasblomquist
Member
 
Location: Ohio

Join Date: Jul 2012
Posts: 68
Default

We scale to 400-1000 samples for amplicon library prep for a flow cell lane on Hiseq and 50-200 samples for miseq easily. Just make sure your barcoding steps are clean and contamination free to prevent "bleed-over" of falsely indexed amplicon products between sample indices. IDT offers Trugrade NGS barcoding primers that work quite well and are worth the investment for zero cross-contamination of reagents. Our libraries are focused panels of 15 to ~100 amplicons per sample.

-Tom
thomasblomquist is offline   Reply With Quote
Old 04-23-2013, 06:27 AM   #5
thomasblomquist
Member
 
Location: Ohio

Join Date: Jul 2012
Posts: 68
Default

Oh, and yes, you need custom bioinformatics/software pipeline to parse out the custom indexes for each sample. The 96 limit is probably Illumina's limit based on their own reagent kits.

-Tom
thomasblomquist is offline   Reply With Quote
Old 04-24-2013, 10:59 AM   #6
james hadfield
Moderator
Cambridge, UK
Community Forum
 
Location: Cambridge, UK

Join Date: Feb 2008
Posts: 221
Default

Dual-indexing should scale cheaply to 384, 1536 and beyond. Uising a pair of indexes to create some thing unique allows just 20 PCR primers to create the 96 used in Nextera for instance. Adding another 20 allows 384 indexes, and increasing to 80 primers (32x i5 & 48x i7) gets you to 1536.
james hadfield is offline   Reply With Quote
Old 04-24-2013, 12:10 PM   #7
JackieBadger
Senior Member
 
Location: Halifax, Nova Scotia

Join Date: Mar 2009
Posts: 381
Default

Quote:
Originally Posted by thomasblomquist View Post
Oh, and yes, you need custom bioinformatics/software pipeline to parse out the custom indexes for each sample. The 96 limit is probably Illumina's limit based on their own reagent kits.

-Tom
jMHC has a GUI and does this well.

Works much faster on the mac, and with large data sets you will need to open the java script from the terminal, allocating more RAM
JackieBadger is offline   Reply With Quote
Old 03-17-2015, 07:00 AM   #8
monia
Junior Member
 
Location: Tunisia

Join Date: Dec 2014
Posts: 4
Default

Hi,
I have 96 samples and i want to design barcodes. How can't do it?
Thax for your help
monia 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 11:18 AM.


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