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Old 08-28-2017, 01:29 PM   #21
Joanna
Junior Member
 
Location: North Carolina

Join Date: Jul 2017
Posts: 9
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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?

Thanks!
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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