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Old 08-28-2017, 12:29 PM   #21
Junior Member
Location: North Carolina

Join Date: Jul 2017
Posts: 9

Hi guys,

I want to post some updates. As people suggested, we ordered fresh primers and made new libraries. Then loaded 6pM with 30% PhiX spike in. And we also doubled the volume of sequencing primers.

This time we had a cluster density of 854 K/mm2 which has improved but PF% was still very low (48.78%) and yield was also low. Aligned PhiX% was just 15% instead of 30%.

My thought is:
Based on the attached thumbnail images, I feel it was over-clustered this time. And since we sequenced low diversity 16S rRNA gene libraries so we had uneven signal distribution for each cycle. This may cause trouble for the camera to correctly identify clusters. So we saw an overall drop in Q30% and "bleeding" (see attached). But this couldn't explain the low PhiX% than expected. And I also noticed that the Q30% of R2 (the first index read) was especially low. Wondering if others met this before? What do you think is the problem?

Attached Images
File Type: png Q30.png (99.2 KB, 15 views)
Attached Files
File Type: pdf run metrics.pdf (141.9 KB, 20 views)
File Type: pdf thumbnails.pdf (1.05 MB, 10 views)
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Old 08-29-2017, 05:11 AM   #22
MU Core
Location: Columbia, Missouri

Join Date: Apr 2008
Posts: 57

Joanna, looks to me as though you've resolved the library issue with the new primers. I would agree that this particular run is over-clustered. We observe similar quality issues when 16S libraries are clustered at this density. Reduce the cluster density and the Q30% will improve. We target cluster density of 650-750 K/mm2 with a 15% spike-in which works well for us on a MiSeq run. I've provided two attachments that display typical results.
Attached Images
File Type: jpg Run metric.JPG (57.0 KB, 14 views)
File Type: jpg Q30plot.JPG (26.1 KB, 15 views)
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Old 08-30-2017, 11:30 AM   #23
Senior Member
Location: CT

Join Date: Apr 2015
Posts: 243

lower than expected phiX indicates that your quant was off. If you loaded your library to get 30% and only could align 15% phiX, that means that your 16S was approximately twice as concentrated as you thought so took up twice as many spots on the flowcell as you planned. Recovering roughly the amount of phiX as you put on is a decent indication that your library quant was correct
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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