HiSeq X different insert size?

[JBKri]

I am about to prepare TruSeq PCR-free libraries for HiSeq X sequencing.
From:

http://support.illumina.com/sequencing/sequencing_instruments/hiseq-x/questions.html

"The supported insert size on the HiSeq X system is 350 bp and 450 bp for TruSeq Nano DNA and TruSeq DNA PCR-Free libraries."

As I see it this phrase could be interpreted in two ways; 350 bp for Nano and 450 bp for PCR-free, or both types of libraries can use either insert size. Anyone knows which it is? If the first; does that mean we MUST make our insert size 450 bp? There is not even a protocol for that in the TruSeq manual.

Jon

[GenoMax]

HiSeq X uses ordered flowcells (like HiSeq 3000/4000). There is a need to control insert size (between 350-450 bp, ideally) with ordered flowcells to avoid generation of optical duplicates due to pad hopping (DNA fragments contaminating multiple wells nearby during the ExAmp procedure).

While you could load longer libraries, you would need to deal with the optical duplicate issue/loss of data downstream.

[JBKri]

HiSeq X uses ordered flowcells (like HiSeq 3000/4000). There is a need to control insert size (between 350-450 bp, ideally) with ordered flowcells to avoid generation of optical duplicates due to pad hopping (DNA fragments contaminating multiple wells nearby during the ExAmp procedure).

While you could load longer libraries, you would need to deal with the optical duplicate issue/loss of data downstream.

Thanks. Does this mean we should do a more stringent size selection than the standard TruSeq two-sided magnetic bead size selection? Do you have any links to protocols for this? The Illumina Custom Protocol selector seems to be broken at the moment.

[GenoMax]

I will let someone else answer that since it is about experimental protocol.

[luc]

The HiSeq 4000, that we are running, is basically the same sequencer as the X sequencer using the same chemistry. Bead based size selection is perfectly sufficient over here.

(if anybody has seen any evidence that pad hooping actually occurs more frequently for longer insert libraries I would be interested).

[JBKri]

The HiSeq 4000, that we are running, is basically the same sequencer as the X sequencer using the same chemistry. Bead based size selection is perfectly sufficient over here.
..

Great, that's just what I was hoping for!