View Single Post
Old 03-08-2017, 06:41 AM   #14
Senior Member
Location: US

Join Date: Apr 2013
Posts: 222

Hi, thanks. Can you explain more about the clustering stage. I don't know much details about HiSeq? Clustering stage -- do you mean it is a step of library building or Bridge amplification?

Originally Posted by Markiyan View Post
Also do not forget the clustering efficiency dependency on insert size.

Basically despite your library having 600bp fragments, they would clusters less efficiently (~10x?) than 200-300 bp fragments present in the sample. As a result one gets a peak on FLASH histogram in the area that is ~1/3x on the rising side of the bell curve produces by bioanalyzer. (You get enrichment of the smaller fragments during the clustering stage.)

PS: with latest iteration of the Illumina instruments (Hiseq4000/NovaSeq) they seem to continue to support libraries with up to 350 bp insert size - Shorter insets give you smaller and brighter (clusters/wells) + less likely to be long enough to jump to neighbouring wells - so can be sequenced on higher densities. As the result we get max 2x150 bp max. support from (Hiseq4000/NovaSeq). If you need 2x250 stick with HiSeq2500 or MiSeq.
SDPA_Pet is offline   Reply With Quote