TruSeq Library Pooling - Balanced Indexes?

[acpetterson]

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]

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.

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Phillip

[NextGenSeq]

Isn't this only a problem with dual indexing?

[pmiguel]

Isn't this only a problem with dual indexing?

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

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Phillip

[snorberg]

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]

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

[pmiguel]

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.

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Phillip