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Old 03-08-2017, 06:41 AM   #14
SDPA_Pet
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Location: US

Join Date: Apr 2013
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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?

Quote:
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.
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