1x500 miseq reads instead of 2x250?

koadman · 03-13-2013, 04:52 AM

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?

JackieBadger · 03-13-2013, 05:34 AM

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

bbeitzel · 03-13-2013, 06:08 AM

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.

GenoMax · 03-13-2013, 07:07 AM

Quote:

Originally Posted by 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.

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.

koadman · 03-15-2013, 02:15 PM

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...

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03-13-2013, 04:52 AM	#1
koadman Member Location: Sydney, Australia Join Date: May 2010 Posts: 65	1x500 miseq reads instead of 2x250? 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?

03-13-2013, 05:34 AM	#2
JackieBadger Senior Member Location: Halifax, Nova Scotia Join Date: Mar 2009 Posts: 381	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

03-13-2013, 06:08 AM	#3
bbeitzel Member Location: Ft. Detrick, MD Join Date: Aug 2008 Posts: 50	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.

03-15-2013, 02:15 PM	#5
koadman Member Location: Sydney, Australia Join Date: May 2010 Posts: 65	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...