Content Comparison

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• Make 1:1000 dilutions of column 1,46,8,11 10 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

• Add 16 ul of Illumina Library Quantification MasterMix to each well:

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• Add 4 ul of template, pool, or standards to each well:

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Results:

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The pool via standard size estimation returned a mean of 42.66 nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 481). 42.66x(452/481) = 40.087 or ~40 nM

Sequencing Test:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

• 1000/Results = ul of Pool to Add

1000/40 = 25uL of Pool to Add

• 1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

1000- 25 = 975 uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 50 pM:

• Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX

• Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.

• Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100

• Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.

Pooling for CU sequencing:

library(tidyverse)
AmfReads <- read.csv("/Users/gregg/Downloads/AMF1_Test_filtermergestats.csv", header = FALSE)
Map <- read.csv("/Users/gregg/Downloads/Bo_Stevens_Sample_Positions.csv", header=TRUE)
Map <- Map[,c(1,2,4)]
Map$order <- c(1:96, 1:92)
AMFmeta <- data.frame(do.call('rbind', strsplit(as.character(AmfReads$V1),'.',fixed=TRUE)))
AmfReads <- cbind(AMFmeta$X2, AmfReads[,2:4])
names(AmfReads) <- c("Sample.ID", "Reads", "Reads2", "Reads3")
AmfReads <- inner_join(AmfReads, Map, by = "Sample.ID")
LowAmf <- AmfReads[ which(AmfReads$Reads < 500),]
write.csv(LowAmf, "/Users/gregg/Downloads/LowAmf.csv", quote = FALSE, row.names = FALSE)

AMF1_Test_filtermergestats.csv Bo_Stevens_Sample_Positions.csv

Since the test sequencing included all samples, we are adjusting pooling for samples that both replicates returned less than 500 reads each. These samples will be pooled at a volume of 20 ul while all others will be pooled at 2 ul. 2 ul from all was combined into one column of strip tubes. 18 ul was added by well pattern above. Tubes were vortexed and centrifuged. 100 ul was added from each strip tube to a 1.5 ml tube which was vortexed and centrifuged before 40 ul was transferred to a tube for shipping.