Nothing
## ---- include = FALSE---------------------------------------------------------
knitr::opts_chunk$set(
collapse = TRUE,tidy = TRUE,
warning=FALSE, message=FALSE,
comment = "##>"
)
## ---- eval = FALSE------------------------------------------------------------
# if (!requireNamespace("BiocManager", quietly = TRUE))
# install.packages("BiocManager")
# BiocManager::install("bambu")
# BiocManager::install("NanoporeRNASeq")
## -----------------------------------------------------------------------------
library(bambu)
test.bam <- system.file("extdata",
"SGNex_A549_directRNA_replicate5_run1_chr9_1_1000000.bam",
package = "bambu")
fa.file <- system.file("extdata",
"Homo_sapiens.GRCh38.dna_sm.primary_assembly_chr9_1_1000000.fa",
package = "bambu")
gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf",
package = "bambu")
bambuAnnotations <- prepareAnnotations(gtf.file)
se <- bambu(reads = test.bam, annotations = bambuAnnotations,
genome = fa.file)
## -----------------------------------------------------------------------------
library(bambu)
library(NanoporeRNASeq)
data("SGNexSamples")
SGNexSamples
library(ExperimentHub)
NanoporeData <- query(ExperimentHub(), c("NanoporeRNA", "GRCh38","BAM"))
bamFiles <- Rsamtools::BamFileList(NanoporeData[["EH3808"]],
NanoporeData[["EH3809"]],NanoporeData[["EH3810"]], NanoporeData[["EH3811"]],
NanoporeData[["EH3812"]], NanoporeData[["EH3813"]])
## -----------------------------------------------------------------------------
# get path to fasta file
genomeSequence <- query(ExperimentHub(), c("NanoporeRNA", "GRCh38","FASTA"))
## -----------------------------------------------------------------------------
library(BSgenome.Hsapiens.NCBI.GRCh38)
genomeSequence <- BSgenome.Hsapiens.NCBI.GRCh38
## -----------------------------------------------------------------------------
gtf.file <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf",
package = "bambu")
annotation <- prepareAnnotations(gtf.file)
## -----------------------------------------------------------------------------
txdb <- system.file("extdata", "Homo_sapiens.GRCh38.91_chr9_1_1000000.gtf",
package = "bambu")
annotation <- prepareAnnotations(txdb)
## -----------------------------------------------------------------------------
data("HsChr22BambuAnnotation")
HsChr22BambuAnnotation
## ---- results = "hide"--------------------------------------------------------
se <- bambu(reads = bamFiles, annotations = HsChr22BambuAnnotation,
genome = genomeSequence, ncore = 1)
## -----------------------------------------------------------------------------
se
## -----------------------------------------------------------------------------
colData(se)$condition <- as.factor(SGNexSamples$cellLine)
## ---- results = "hide"--------------------------------------------------------
seUnextended <- bambu(reads = bamFiles,
annotations = HsChr22BambuAnnotation,
genome = genomeSequence, discovery = FALSE)
## -----------------------------------------------------------------------------
seUnextended
## ---- fig.width = 8, fig.height = 6-------------------------------------------
library(ggplot2)
plotBambu(se, type = "heatmap")
## ---- fig.width = 8, fig.height = 6-------------------------------------------
plotBambu(se, type = "pca")
## ---- fig.width = 8, fig.height = 10------------------------------------------
plotBambu(se, type = "annotation", gene_id = "ENSG00000099968")
## -----------------------------------------------------------------------------
seGene <- transcriptToGeneExpression(se)
seGene
## ---- fig.width = 8, fig.height = 6-------------------------------------------
colData(seGene)$groupVar <- SGNexSamples$cellLine
plotBambu(seGene, type = "heatmap")
## -----------------------------------------------------------------------------
save.dir <- tempdir()
writeBambuOutput(se, path = save.dir, prefix = "NanoporeRNASeq_")
## -----------------------------------------------------------------------------
save.file <- tempfile(fileext = ".gtf")
writeToGTF(rowRanges(se), file = save.file)
## -----------------------------------------------------------------------------
library(DESeq2)
dds <- DESeqDataSetFromMatrix(round(assays(seGene)$counts),
colData = colData(se),
design = ~ condition)
dds.deseq <- DESeq(dds)
deGeneRes <- DESeq2::results(dds.deseq, independentFiltering = FALSE)
head(deGeneRes[order(deGeneRes$padj),])
## -----------------------------------------------------------------------------
summary(deGeneRes)
## ---- fig.width = 8, fig.height = 6-------------------------------------------
library(apeglm)
resLFC <- lfcShrink(dds.deseq, coef = "condition_MCF7_vs_K562", type = "apeglm")
plotMA(resLFC, ylim = c(-3,3))
## -----------------------------------------------------------------------------
library(DEXSeq)
dxd <- DEXSeqDataSet(countData = round(assays(se)$counts),
sampleData = as.data.frame(colData(se)),
design = ~sample + exon + condition:exon,
featureID = rowData(se)$TXNAME,
groupID = rowData(se)$GENEID)
dxr <- DEXSeq(dxd)
head(dxr)
## ----fig.width = 8, fig.height = 6--------------------------------------------
plotMA(dxr, cex = 0.8 )
## ---- eval = FALSE------------------------------------------------------------
# se <- bambu(reads = bamFiles, rcOutDir = "./bambu/",
# annotations = annotaiton,
# genome = genomeSequence)
## ---- eval = FALSE------------------------------------------------------------
# bambu(reads, annotations, genome,
# opt.discovery = list(min.readCount = 5))
## ---- eval = FALSE------------------------------------------------------------
# bambu(reads, annotations, genome,
# opt.discovery = list(min.sampleNumber = 5))
## ---- eval = FALSE------------------------------------------------------------
# bambu(reads, annotations, genome,
# opt.discovery = list(min.readFractionByGene = 0.1))
## ---- eval = FALSE------------------------------------------------------------
# bambu(reads, annotations, genome, opt.em = list(bias = FALSE))
## ---- eval = FALSE------------------------------------------------------------
# bambu(reads, annotations, genome, ncore = 8)
## -----------------------------------------------------------------------------
sessionInfo()
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