RNA-seq multiplexing

[sppearce]

I'm currently planning my first large RNA-seq experiment and was unsure about large-scale multiplexing.

I will have a total of 48 libraries and one option would be run 12 lanes with four libraries each. However, if it is possible, a better alternative would be to run all 48 libraries in each lane because 1) We could run an initial test lane to check quality and coverage and 2) we can easily scale up to run extra lanes to add required coverage.

With the Tru-seq RNA kit, they provide only 6 adapters per kit (12 with A and B kits combined) so I was wondering if anybody had any experience in making their own adapters, either using the older "in line" adapters or the newer method used by the Truseq kit with the separate seq read to determine the barcode. If so, are the results comparable to those using the commercial adapters and can they be easily substituted into the Truseq kit?

Any other alternative strategies or advice on the best way forward here would be greatly appreciated.

Thanks!

Stephen

[pmiguel]

I completely agree with you. The only kit I see that has all 48 TruSeq adapters is the small RNA kit.

I guess I come to the Illumina world late, but their handling of bar codes seems really clunky. Seems absolutely obvious that 48 or 96 bar codes would be great. Here are some reasons:

(1) There is no down side. Is there? Why is having 6 adapters in a 48 library kit better than 48 adapters?

(2) Titration becomes easy. If you are a little unsure of the right amount to add for optimum cluster densities for each library, just run a lane with every single library you will use in the next X runs. Makes no amplification libraries pretty easy to deal with.

(3) v3 chemistry easily gives you >20 billion bases of sequence per HiSeq lane. The value of going >100X in coverage on a resequencing or de novo genome sequence is questionable. So for any genome less 10 megabases or so, you want to run more than 12 libraries at a time.

I was planning to order oligos for indexes 13-24 and see how they work. But why Illumina doesn't just sell a 48 or 96 index TruSeq adapter kit is beyond me.

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Phillip