I am running some bisulfite converted amplicon-seq libraries (Fluidigm Access Array) on a MiSeq with version2 300-cycle reagents without PhiX (I will be spiking in 5% PhiX in future runs). As this is a low diversity pool and it has been bisulfite treated, I have been expecting lower than normal quality scores, however, I am seeing a rather low %PF. I have run two pools so far which have the following run metrics:
cluster density %PF AVG%Q30
Run1 935K/mm2 75.93% 88.84%
Run2 695K/mm2 65.4% 85.36%
I am not sure whether I should be concerned about the low %PF or whether it is expected for bisulfite converted amplicon libraries. Is there anything I can do to help increase the %PF? Can I still continue with analysing the resulting FASTQs from these runs or is that a bad idea?
Any help would be greatly appreciated.
Edit:
I checked the optic images and they not overclustered.
The first 22 bp of my insert is the Fluidigm sequencing primer and because the %PF is determined after the first 25 cycles the machine might 'think' all the libraries/clusters are homogenous fooling it into determining the run as being overclustered and giving a low %PF. Does this reasoning seem like an explanation?
Edit2:
Yield of the first run was 5.28Gbp and 4.07Gbp and Illumina advertise the maximum v2 yield as 5.1Gb, suggesting most clusters are in actuality passing filter.
cluster density %PF AVG%Q30
Run1 935K/mm2 75.93% 88.84%
Run2 695K/mm2 65.4% 85.36%
I am not sure whether I should be concerned about the low %PF or whether it is expected for bisulfite converted amplicon libraries. Is there anything I can do to help increase the %PF? Can I still continue with analysing the resulting FASTQs from these runs or is that a bad idea?
Any help would be greatly appreciated.
Edit:
I checked the optic images and they not overclustered.
The first 22 bp of my insert is the Fluidigm sequencing primer and because the %PF is determined after the first 25 cycles the machine might 'think' all the libraries/clusters are homogenous fooling it into determining the run as being overclustered and giving a low %PF. Does this reasoning seem like an explanation?
Edit2:
Yield of the first run was 5.28Gbp and 4.07Gbp and Illumina advertise the maximum v2 yield as 5.1Gb, suggesting most clusters are in actuality passing filter.
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