Page Comparison

Alex Buerkle Linda van Diepen

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I think we need to either do a larger experiment prior to sending out NovaSeq5. Or, we pool NovaSeq5 as normal, qPCR the first 72 samples singly from 10? plates and then use the sequencing data from it with the qPCR data to decide the pooling standard moving forward. I would advocate for option B.

We are going to add added absorbance into the mix as a cheaper and quicker tool for normalized pooling. We will get got absorbance readings for these same products and do the same comparison to readsdid the same comparison to reads.

#Read in the absorbance data
AbsLVD <- read_xlsx("/Volumes/Macintosh HD/Users/gregg/Downloads/5LVD5_16S_ITS.xlsx", sheet = "Summary", skip = 2)
AbsLVD <- AbsLVD[, c(3,11)]
AbsDG <- read_xlsx("/Volumes/Macintosh HD/Users/gregg/Downloads/5DG5_16S_ITS.xlsx", sheet = "Summary", skip = 2)
AbsDG <- AbsDG[, c(3,11)]
AbsMC <- read_xlsx("/Volumes/Macintosh HD/Users/gregg/Downloads/LRII_LRIII_MC.xlsx", sheet = "Summary", skip = 2)
AbsMC <- AbsMC[, c(3,11)]
AbsLRIII <- rbind(AbsLVD, AbsDG, AbsMC)
colnames(AbsLRIII) <- c("samplename", "NgPerUl")

LRIIIReads <- left_join(LRIIIReads, AbsLRIII, by = "samplename")

#First lets check the relationship to reads
ggplot(LRIIIReads, aes(NgPerUl, reads, color=plate, shape=plate))+
  geom_point(show.legend = TRUE)+
  geom_smooth(method='lm', formula= y~x)+
  stat_regline_equation(label.y = 200000, aes(label = ..eq.label..)) +
  stat_regline_equation(label.y = 150000, aes(label = ..rr.label..))+
  facet_wrap(~plate)

Image Added

There is still a relationship here. We will proceed with 1 column of qPCR as a check on sequence-able state, and use absorbance to adjust for less read disparity.

We will perform absorbance checks, compile the data, and then sort out the best program for normalization. Probably, we will normalize to around 10 ng/ul. 1 nM is the minimum library concentration for a NovaSeq run. From the data here, 1.44 nM ~ 4 ng/ul. We would like to operate under a larger margin of error.

Is there a relationship between reads and qPCR?

ggplot(LRIIIReads, aes(NgPerUl, mean, color=plate, shape=plate))+
  geom_point(show.legend = TRUE)+
  geom_smooth(method='lm', formula= y~x)+
  stat_regline_equation(label.y = 125, aes(label = ..eq.label..)) +
  stat_regline_equation(label.y = 100, aes(label = ..rr.label..))+
  facet_wrap(~plate)

Image Added

Excluding the Mock Community samples, there is a strong relationship between absorbance and qPCR. The odd MC results may be a result of the small Data Set, their larger than average contribution to the data set, and their very low relative complexity.

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