lots of overlapping reads - Pair-end library from Nextera

[clariet]

We have done a couple of run (2X150 and 2X250) of Miseq on bacteria genome sequencing. They all have a high overlapping rate (>80%).

We used Nextera XT for sample prep. I am interested to see any other users of Nextera and what your experience is on insert length and how we could increase insert length in order to reduce overlapping reads.

Thanks in advance.
Clariet

[Torst]

Yes this is unfortunately very common. Library prep is very sensitive to timings and amounts.

Read about Nick's experiences here: http://pathogenomics.bham.ac.uk/blog...era-libraries/

[cliffbeall]

We had a lot of overlap even with 100 bp paired end and an older Nextera. I think the average insert was around 150. Like the pathogenomics blog post I used Trimmomatic to remove the adapters - it has a sensible way of doing it. Also like the post I got pretty good assemblies of bacteria.

I don't know of a sure way to increase insert size, but you might look at reducing the ratio of transposase to target DNA. I would give Epicentre a call - they have been helpful to me in the past.