MiSeq Nextera XT expected output?

[JPC]

Hello all, how many raw reads am I likely to get from a single MiSeq run with a Nextera XT sample? .....am I being too cynical by assuming that as this is a protocol designed for set-up speed it won't get up to the 30-34M PF reads that I would expect for a normal Paired End run?

JPC

[RCJK]

I think it is possible to obtain that amount of reads. Maybe slightly less, but not way off, since it's a bit more difficult to get optimised loading due to a wide range of fragment sizes.

[koadman]

Hi JPC, in our experience when using the protocol's suggested dilution into hybridization buffer at the last step, the number of reads varies widely by sample type. I'm not sure whether this is due to input material quantitation differences or something else. The protocol suggests the dilution can be changed and presumably this will affect read count. Our group plans to try it this week.

[GenoMax]

PS: Info below is not specific for Nextera runs but a general observation with MiSeq runs.

MiSeq runs are affected (in terms of yield and sequence quality) to a greater extent if your samples have reduced nucleotide diversity (e.g. 16S). There are other threads (e.g. http://seqanswers.com/forums/showthr...t=20455&page=3) that have information about this known observation specially for long read lengths (2 x 250 bp).

If you are sequencing normal genomic DNA then you can reasonably expect to recover standard number of reads. If your samples have reduced nucleotide diversity then the number of reads passing filter can potentially experience a significant drop.