Illumina Long ReadKit (Moleculo) on MiSeq

[NGSsequencer]

Has anyone taken the moleculo kit and run on the 250bp runs on the Miseq and tried the assembly after? I'd like to know if it would work. On the Illumina website only up to the 2500 is supported, but I figured doing this on the longer reads on the MiSeq would enhance constructing the long reads and phasing downstream.

thoughts, experiences?

thanks

[nucacidhunter]

Assembly of short reads for obtaining sequences of input long fragments is not based on overlap only. Longer reads may cause a few issues:
1) Assembly of short reads to reconstruct the original 10 kb fragments is currently only possible on BaseSpace Apps. Those Apps has been designed to process supported read length and may be unable to process longer reads.
2) Minimum and maximum data supported by Apps are 30 and 115 GB, respectively. Running on MiSeq will not meet the minimum required data.
3) A sample sheet is required to process data on BaseSpace apps. Considering the data is fed live to BaseSpace it will be very difficult if not impossible to feed data from multiple MiSeq runs to meet the minmum data requirement.
4) Regardless of read length the maximum synthetic read length will be the input length (~10 kb), so whatever one does any length beyond input fragment length will be assembly artefact.

I think Illumina has not completely optimised this kit (at least size-selection step) indicated by presence of lots of shorter synthetic reads than size-selected 10 kb fragments in their respected data sheets. One way to improve this would be alternative size-selection method to reduce number of shorter than target fragments that go through PCR amplification step.

Here is a link to illumina’s current analysis workflow for phasing: http://supportres.illumina.com/docum...15055852-a.pdf