1x500 miseq reads instead of 2x250?

[koadman]

Well of course I was hoping not to be the sacrificial lamb, but if nobody else has tried this and able to share data I guess I will take one for the team! Now just need to find a willing pair of lab hands here in Sydney...

[GenoMax]

We've done several 1x300 runs with 300 cycle kits, and there is a noticeable dropoff in quality after ~base 250 on good runs. I can't imagine that a 1x500 run would give you more than ~300 bases of usable data.

The 1x300 runs you are referring to were likely done with v.1 kits?

It is great if koadman is willing to sacrifice a v.2 kit and enlighten all on two things: if the 1 x 500 will work and where the drop-off in qualities can be expected to start.

[bbeitzel]

We've done several 1x300 runs with 300 cycle kits, and there is a noticeable dropoff in quality after ~base 250 on good runs. I can't imagine that a 1x500 run would give you more than ~300 bases of usable data.

[JackieBadger]

I dont see how it would. If the sum of your amplicon is shorter than that of your paired ends you can join them based on a region of overlap

[koadman]

A while back ECO posted some great info on a 1x300 miseq run. Has anybody tried a similar run with an upgraded instrument to achieve a 500 nt read? Will the software permit this? How do the base qualities trail off?