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Old 05-11-2017, 08:06 AM   #1
Location: uk

Join Date: Mar 2015
Posts: 14
Default Acceptable Passing Filter (%PF)

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.

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?

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.

Last edited by dross11; 05-11-2017 at 10:00 AM. Reason: reinstated last edit
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Old 05-11-2017, 09:00 AM   #2
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spiking in phiX should increase your pf% but those numbers aren't horrible given you didn't spike in. For my amplicon runs, I spike in 15% phiX and usually see ~80-90% pf

No reason not to use this data, what's passed filter and been demultiplexed should be fine
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.
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Old 05-12-2017, 12:33 AM   #3
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Do you need the fluidigm seq primer for anything in the downstream processing? If not, I would suggest to run 10-20 dark cycles (depends if the primer seq is really at the start of every fragment) in the sequencing protocol. If you have a normal seq complexity after the primer, you will not run into any problems in terms of cluster identification.
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Old 05-12-2017, 09:24 AM   #4
Location: uk

Join Date: Mar 2015
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I performed a run today. I spiked in 5% PhiX and the %PF is at 73.49% and quality is at ~94% with a cluster density of ~1100K/mm2. I'll try 15% next time but I am fairly certain the lowish %PF is due to the sequencing primers presence within my insert. I will also look into performing 15 dark cycles next time round too (this is really very clever).

Thank you for your help and reassurance

Last edited by dross11; 05-12-2017 at 09:26 AM. Reason: More info
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