TruSeq Library Pooling - Balanced Indexes?

acpetterson · 04-18-2012, 09:17 AM

Hi:

I typically pool 6 TruSeq libraries with P5/P7 adapter pairs (2 4 5 6 7 12) per lane, as per Illumina's recommended pooling for balanced indexes.

I need to pool 8 libraries, and am having a difficult time finding the pooling strategy recommended for this number. Does anyone have experience with this strategy? In my notes from Illumina, I only have recommendations for 2, 3, 4, and 6 pooled libraries.

Thanks,

ap

pmiguel · 04-18-2012, 12:00 PM

Don't sweat it. Above 6 indexes you have to work to create an imbalance that would throw the instrument software off. If you are feeling paranoid, use your balance pool of 6 indexes and spike in any other 2 at about the same molar amount.

--
Phillip

NextGenSeq · 04-25-2012, 02:57 PM

Isn't this only a problem with dual indexing?

pmiguel · 04-26-2012, 04:55 AM

Quote:

Originally Posted by NextGenSeq

Isn't this only a problem with dual indexing?

Why would it be? (I haven't done any dual indexing runs yet.)

--
Phillip

snorberg · 01-17-2013, 01:15 PM

You are free to use whatever indexes you like.

Refer to Illumina's "Low-Plex Pooling Guidelines with TruSeq DNA or RNA Sample Prep v2". For pooling 5-11 libraries, it recommends using one of the 4-plex options + any other indexes.

Best, Steve

share · 01-19-2013, 01:59 AM

Why don't you use illumina's experiment manager? We use it always.

pmiguel · 01-25-2013, 05:55 AM

Quote:

Originally Posted by share

Why don't you use illumina's experiment manager? We use it always.

I tried it out when first launched. I could feel my brain starting to bleed after a few minutes. I stopped at that point, hopefully before a great deal of irreversible damage was done.

If I were starting from scratch, I would probably use it. But we already have our own methods to generate sample sheets tied in to our sample tracking software.

--
Phillip

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04-18-2012, 09:17 AM	#1
acpetterson Junior Member Location: Irvine Join Date: Feb 2012 Posts: 1	TruSeq Library Pooling - Balanced Indexes? Hi: I typically pool 6 TruSeq libraries with P5/P7 adapter pairs (2 4 5 6 7 12) per lane, as per Illumina's recommended pooling for balanced indexes. I need to pool 8 libraries, and am having a difficult time finding the pooling strategy recommended for this number. Does anyone have experience with this strategy? In my notes from Illumina, I only have recommendations for 2, 3, 4, and 6 pooled libraries. Thanks, ap

04-18-2012, 12:00 PM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Don't sweat it. Above 6 indexes you have to work to create an imbalance that would throw the instrument software off. If you are feeling paranoid, use your balance pool of 6 indexes and spike in any other 2 at about the same molar amount. -- Phillip

04-25-2012, 02:57 PM	#3
NextGenSeq Senior Member Location: USA Join Date: Apr 2009 Posts: 482	Isn't this only a problem with dual indexing?

01-17-2013, 01:15 PM	#5
snorberg Junior Member Location: San Diego, CA Join Date: Jan 2013 Posts: 5	You are free to use whatever indexes you like. Refer to Illumina's "Low-Plex Pooling Guidelines with TruSeq DNA or RNA Sample Prep v2". For pooling 5-11 libraries, it recommends using one of the 4-plex options + any other indexes. Best, Steve

01-19-2013, 01:59 AM	#6
share Member Location: india Join Date: Mar 2011 Posts: 14	Why don't you use illumina's experiment manager? We use it always.