Here are some observations for clustering density with PCR-free DNA preps, and I'm wondering if anyone else has observed the same? On several PCR-free preps, we run the samples at our usual concentration, and get ~60-70% of the expected yield. This probably isn't unexpected, but when we increase the concentration on subsequent runs, the cluster density hits a ceiling and then starts decreasing (sometimes dramatically). Here are some numbers from a HiSeq2000 high output flowcell run with three different concentrations of the same library:
Library at 8 pM: 177M total reads/164M PF reads
Same library at 10 pM: 171M total reads/160M PF reads
Same library at 12 pM: 141M total reads/134M PF reads
The cluster plots for the higher concentrations don't look like an overclustered lane (with a lower PF rate). Images look fine....just lower density.
I assume this is because of the library molecules in the mix with only one adaptor ligated on the ends, not both, interfering with cluster formation.
Any ideas/comments? Is this too much DNA going into the ligation step? Any other solutions?
Library at 8 pM: 177M total reads/164M PF reads
Same library at 10 pM: 171M total reads/160M PF reads
Same library at 12 pM: 141M total reads/134M PF reads
The cluster plots for the higher concentrations don't look like an overclustered lane (with a lower PF rate). Images look fine....just lower density.
I assume this is because of the library molecules in the mix with only one adaptor ligated on the ends, not both, interfering with cluster formation.
Any ideas/comments? Is this too much DNA going into the ligation step? Any other solutions?
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