Created
December 16, 2019 12:33
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Example of running dif expr analysis with outputs from salmon
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| library(GenomicRanges) | |
| library(DESeq2) | |
| library(data.table) | |
| library(dplyr) | |
| library(stringr) | |
| library(gplots) | |
| library(tximport) | |
| library(openxlsx) | |
| # Genes were taken from flybase I think, selecting only GENEs from full annotation | |
| # E.g. ftp://ftp.flybase.net/releases/FB2019_05/dmel_r6.30/gff/dmel-all-r6.30.gff.gz | |
| genes <- fread("~/Database/dmel/Annotations/dmel-gene-r5.57.gff") %>% | |
| dplyr::select(c(1, 4, 5, 7, 9)) %>% | |
| setNames(c("chr", "start", "end", "strand", "attr")) %>% | |
| mutate(id = sub('^ID=(FBgn[0-9]+);.*', '\\1', attr), | |
| chr = paste0("chr", chr), gene_name = sub('.*Name=([^;]+);.*', '\\1', attr), | |
| tss = ifelse(strand == "+", start, end)) %>% | |
| dplyr::select(-attr) | |
| genes.gr <- GRanges( | |
| seqnames = Rle(genes$chr), | |
| ranges = IRanges( | |
| start = genes$start, | |
| end = genes$end, | |
| names = genes$gene_name | |
| ) | |
| ) | |
| # IMPORT SALMON OUTPUT | |
| files <- paste0("/home/artem/IMG/Projects/HP1.ovaries/rnaseq/salmon_out/190325defset/170914_HSGA.Shevelev_RNA_2017.", | |
| c("control1.trim", "control2.trim", "KD1.trim", "KD2.trim")) | |
| files <- file.path(files, "quant.genes.sf") | |
| names(files) <- c("WT1", "WT2", "HP1KD1", "HP1KD2") | |
| txi.hp1 <- tximport(files, "salmon", txOut = T) | |
| head(txi.hp1$counts) | |
| # DESEQ2 | |
| samples <- data.frame(row.names = names(files), conditions = c("wt", "wt", "kd", "kd"), orig = "evrogen") | |
| ddsTxi <- DESeqDataSetFromTximport(txi.hp1, colData = samples, design =~ conditions) | |
| keep <- rowSums(counts(ddsTxi)) >= 10 | |
| ddsTxi <- ddsTxi[keep,] | |
| ddsTxi$conditions <- relevel(ddsTxi$conditions, ref = "wt") | |
| dds <- DESeq(ddsTxi) | |
| res <- results(dds) | |
| resultsNames(dds) | |
| resLFC <- lfcShrink(dds, coef = "conditions_kd_vs_wt", type = "apeglm", | |
| lfcThreshold = 1) | |
| pdf("./plots/MA.plot.pdf") | |
| DESeq2::plotMA(resLFC, alpha = 0.05, ylim=c(-6, 6)) | |
| dev.off() | |
| summary(res, alpha = 0.05) | |
| res.df <- as.data.frame(res) %>% mutate(id = rownames(res)) | |
| res.df <- merge(genes, res.df, by = "id", all.y = T) | |
| write.xlsx(res.df, file = "./tables/DESeq2.hp1kd.results.xlsx", dec = ",") | |
| # res.df %>% filter(is.na(gene_name)) %>% View | |
| res.df.tss.gr <- makeGRangesFromDataFrame(res.df %>% | |
| mutate(start = tss, | |
| end = tss) %>% | |
| filter(-tss, padj < 0.05), | |
| keep.extra.columns = T) |
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