Library Preparation
Sequencing
Bioinformatics Analysis
Image adapted from: Wang, Z., et al. (2009), Nature Reviews Genetics, 10, 57–63.
November 2024
Library Preparation
Sequencing
Bioinformatics Analysis
Image adapted from: Wang, Z., et al. (2009), Nature Reviews Genetics, 10, 57–63.
Picard Tools:
library(tximport) txi <- tximport(salmon_files, type = "salmon", tx2gene = tx2gene) str(txi)
## List of 4 ## $ abundance : num [1:35896, 1:12] 20.381 0 1.966 1.059 0.949 ... ## ..- attr(*, "dimnames")=List of 2 ## .. ..$ : chr [1:35896] "ENSMUSG00000000001" "ENSMUSG00000000003" "ENSMUSG00000000028" "ENSMUSG00000000037" ... ## .. ..$ : chr [1:12] "SRR7657878" "SRR7657881" "SRR7657880" "SRR7657874" ... ## $ counts : num [1:35896, 1:12] 1039 0 65 39 8 ... ## ..- attr(*, "dimnames")=List of 2 ## .. ..$ : chr [1:35896] "ENSMUSG00000000001" "ENSMUSG00000000003" "ENSMUSG00000000028" "ENSMUSG00000000037" ... ## .. ..$ : chr [1:12] "SRR7657878" "SRR7657881" "SRR7657880" "SRR7657874" ... ## $ length : num [1:35896, 1:12] 2905 541 1884 2100 480 ... ## ..- attr(*, "dimnames")=List of 2 ## .. ..$ : chr [1:35896] "ENSMUSG00000000001" "ENSMUSG00000000003" "ENSMUSG00000000028" "ENSMUSG00000000037" ... ## .. ..$ : chr [1:12] "SRR7657878" "SRR7657881" "SRR7657880" "SRR7657874" ... ## $ countsFromAbundance: chr "no"
rlogCounts <- rlog(filtCounts)
txiObj <- readRDS("RObjects/txi.rds") sampleinfo <- read_tsv("data/samplesheet_corrected.tsv", col_types="cccc") %>% mutate(Status = fct_relevel(Status, "Uninfected"))
model <- as.formula(~ TimePoint + Status + TimePoint:Status)
ddsObj <- DESeqDataSetFromTximport(txi = txiObj, colData = sampleinfo, design = model)
keep <- rowSums(counts(ddsObj) > 5 ddsObj <- ddsObj[keep,]
ddsObj <- DESeq(ddsObj)
results.day11 <- results(ddsObj, name="Status_Infected_vs_Uninfected", alpha=0.05) results.day33 <- results(ddsObj, contrast = list(c("Status_Infected_vs_Uninfected", "TimePointd33.StatusInfected")), alpha=0.05)