Content Comparison

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• 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.