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Lovro 06-20-2016 02:32 AM

Truseq libray spike-in into Nextera library run

In our lab we routinly perform exome sequencing with Nextera kits on Mi/HiSeq platforms.
In my experiment I want to sequence libraries made with Truseq DNA PCR-free LT kit and enriched with custom IDT xGen probes.
Since the targeted sequence is relatively small, or me it would be the cheapest to add/spike this library into the runs we perform on regular basis (Nextera).

I found this on Illumina's FAQ:
Can I run TruSeq HT libraries with Nextera libraries on the same lane or same flow cell? Are the indices the same or different?

Illumina does not support, and strongly advises against, running libraries prepared by different sample prep kits in the same lane of a flow cell. We do support running libraries prepared by different sample prep kits in different lanes of the same flow cell or spiking in llumina PhiX control library in the same lane as any user-prepared libraries. If different library types are run in different lanes, Dual Index Recipes and the Dual Index Primer Box must be used. The indices between TruSeq HT and Nextera are unique and not shared. Please see appropriate sample prep user guides for index sequences.

It is for the HT version of the kit, but it also says that dual indexing is necessary. Tru-seq pcr-free LT kits are single index. Does that mean I absolutely can't do the spike in? What are your experiences?

If anybody has any experience with mixing libraries made by different protocols on Illumina platroftm, can he/she please comment.

Thank you!

Markiyan 06-20-2016 06:18 AM

As long as the sequencing/index primers are compatible, it is possible to mix.
Dear Lovro,

In principle, as long as the sequencing and index primers are compatible, it is possible to mix libraries in the same lane, but one should be extremely careful with the insert size distribution (make sure there are no small fragments/adapter dimers in any input libs).

Also make sure you allow enough cycles on the index reads (if using 6bp and 8bp indexed reads) use 8 cycles for index.
Demultiplexing can become quite tricky (esp if you have different length indexes), but is doable with manual samplesheet.csv editing.

See the following thread for more info.

I've demultiplexed a couple of runs with Truseq PCR-free + nextera MatePair and a more tricky case of Nextera Shotgun with Nextera MatePair runs.

But if you've got enough samples - do a separate runs if possible - usually it gives better and more consistent performance.

thermophile 06-20-2016 08:24 AM

I've run truseq lt with my homebrew dual indexed amplicons. It works fine but I'm pooling very different things and expect to have some wildly different pooling effeciencies. For the sample sheet you'll need to add NN to the reverse index, and NNNNNNNN for the forward (of course after checking that none of your nextera indexes start with the 6pb of the truseq index)

Lovro 06-22-2016 12:36 AM

Thank you both for your answers and a link!
As long as it can be done I will try it. I will get back with more questions when I get my reagents and prepare and enrich the libraries.

Thank you!

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