Package 'APAlyzer'

APABox, APA RED Box plotting

Description

APA RED Box plotting

Usage

APABox (df, xlab = "APAreg", ylab = "RED",
						plot_title = NULL)

Arguments

df	a dataframe of APAdiff output
xlab	lable of x-axis, default is 'APAreg'
ylab	lable of y-axis, default is 'RED'
plot_title	Main title of plot

Value

The function APABox return a Box plot.

Author(s)

Ruijia Wang

Examples

library("TBX20BamSubset")
    library("Rsamtools")
    flsall = getBamFileList()
    extpath = system.file("extdata",
    "mm9_TBX20.APAout.RData", package="APAlyzer")
    load(extpath)
    sampleTable1 = data.frame(samplename = c(names(flsall)),
        condition = c(rep("NT",3),rep("KD",3)))
    sampleTable2 = data.frame(samplename = c("SRR316184","SRR316187"),
        condition = c("NT","KD"))
    ## 3'UTR APA plot
    test_3UTRmuti=APAdiff(sampleTable1,DFUTRraw,
    conKET='NT',trtKEY='KD',PAS='3UTR',CUTreads=0)
	UTR_APA_PLOTBOX=APABox(test_3UTRmuti, plot_title='3UTR APA')

    ## IPA plot
    test_IPAmuti=APAdiff(sampleTable1,IPA_OUTraw,
    conKET='NT',trtKEY='KD',PAS='IPA',CUTreads=0)
	IPA_PLOTBOX=APABox(test_IPAmuti, plot_title='IPA')

---

APAdiff, calculate delta relative expression (RED) and statistics significance between two sample groups

Description

Calculate delta relative expression (RED) and statistics significance between two sample groups.

Usage

APAdiff(sampleTable,mutiraw, conKET='NT',
    trtKEY='KD', PAS='3UTR', CUTreads=0, p_adjust_methods="fdr", MultiTest='unpaired t-test')

Arguments

sampleTable	a dataframe of sample table containing 8 colmuns for Intronic PASs: 'samplename','condition'
mutiraw	a dataframe output obtained using either PASEXP_3UTR or PASEXP_IPA
conKET	the name of control in the sampletable, default is 'NT'
trtKEY	the name of control in the sampletable, default is 'KD'
PAS	type of PAS analyzed, either '3UTR' or 'IPA', default is '3UTR'
CUTreads	reads cutoff used for the analysis, default is 0
p_adjust_methods	p value correction method, the method can be "holm", "hochberg", "hommel", "bonferroni", "BH", "BY","fdr", "none", default is "fdr"
MultiTest	statistics testing method for muti-replicates designs, the method can be "unpaired t-test", "paired t-test", "ANOVA", default is "unpaired t-test"

Value

The function APAdiff return a dataframe containning RED, pvalue and regulation pattern (UP, DN or NC) for either each gene (3'UTR APA) or each PAS (IPA).

Author(s)

Ruijia Wang

Examples

library("TBX20BamSubset")
    library("Rsamtools")
    flsall = getBamFileList()
    extpath = system.file("extdata",
    "mm9_TBX20.APAout.RData", package="APAlyzer")
    load(extpath)
    sampleTable1 = data.frame(samplename = c(names(flsall)),
        condition = c(rep("NT",3),rep("KD",3)))
    sampleTable2 = data.frame(samplename = c("SRR316184","SRR316187"),
        condition = c("NT","KD"))
    ## Analysis 3'UTR APA between KD and NT group using muti-replicates
    test_3UTRmuti=APAdiff(sampleTable1,DFUTRraw,
    conKET='NT',trtKEY='KD',PAS='3UTR',CUTreads=0,p_adjust_methods="fdr",MultiTest='unpaired t-test')

    ## Analysis 3'UTR APA between KD and NT group without replicates
    test_3UTRsing=APAdiff(sampleTable2,DFUTRraw,
    conKET='NT',trtKEY='KD',PAS='3UTR',CUTreads=0,p_adjust_methods="fdr")

    ## Analysis IPA between KD and NT group
    test_IPAmuti=APAdiff(sampleTable1,IPA_OUTraw,
    conKET='NT',trtKEY='KD',PAS='IPA',CUTreads=0,p_adjust_methods="fdr",MultiTest='unpaired t-test')

    ## Analysis IPA between KD and NT group without replicates
    test_IPAsing=APAdiff(sampleTable2,IPA_OUTraw,
    conKET='NT',trtKEY='KD',PAS='IPA',CUTreads=0,p_adjust_methods="fdr")

---

APAVolcano, APA Volcano plotting

Description

APA Volcano plotting

Usage

APAVolcano (df, Pcol = "pvalue",PAS='3UTR',
						top = -1, markergenes = NULL,
						y_cutoff = 0.05,xlab = "RED", ylab = "-Log10(P-value)",
						PAScolor = c("gray80", "red", "blue"),
						alpha = 0.75, plot_title = NULL,
						width = 4, height = 2.5)

Arguments

df	a dataframe of APAdiff output
Pcol	p-value column used to for y-axis of volcano plot, default is 'pvalue'
top	number of genes/IPA to label in the plot, default is -1, which don't lable top genes, user can set it >0, e.g., top = 5
markergenes	a set of genes to label in the plot
PAS	type of PAS analyzed, either '3UTR' or 'IPA', default is '3UTR'
y_cutoff	y cutoff line, default is 0.05
xlab	lable of x-axis, default is 'RED'
ylab	lable of y-axis, default is '-Log10(P-value)'
PAScolor	dot color for 'NC','UP' and 'DN' gene/IPAs, default is "gray80", "red", and "blue"
alpha	alpha of the dot, default is 0.75
plot_title	Main title of plot
width	width of the dot, default is 4
height	height of the dot, default is 2.5

Value

The function APAVolcano return a Volcano plot.

Author(s)

Ruijia Wang

Examples

library("TBX20BamSubset")
    library("Rsamtools")
    flsall = getBamFileList()
    extpath = system.file("extdata",
    "mm9_TBX20.APAout.RData", package="APAlyzer")
    load(extpath)
    sampleTable1 = data.frame(samplename = c(names(flsall)),
        condition = c(rep("NT",3),rep("KD",3)))
    sampleTable2 = data.frame(samplename = c("SRR316184","SRR316187"),
        condition = c("NT","KD"))
    ## 3'UTR APA plot
    test_3UTRmuti=APAdiff(sampleTable1,DFUTRraw,
    conKET='NT',trtKEY='KD',PAS='3UTR',CUTreads=0)
	UTR_APA_PLOT=APAVolcano(test_3UTRmuti, PAS='3UTR', Pcol = "pvalue", top=5, plot_title='3UTR APA')

    ## IPA plot
    test_IPAmuti=APAdiff(sampleTable1,IPA_OUTraw,
    conKET='NT',trtKEY='KD',PAS='IPA',CUTreads=0)
	IPA_PLOT=APAVolcano(test_IPAmuti, PAS='IPA', Pcol = "pvalue", top=5, plot_title='IPA')

---

download_testbam, download bam files of mouse testis and heart

Description

download bam files of mouse testis and heart

Usage

download_testbam()

Value

The function download_testbam download test data bam files.

Author(s)

Ruijia Wang

Examples

download_testbam()

---

GENEXP_CDS, count reads mapped to CDS regions and calculate TPM for coding gene

Description

Map reads to CDS regions and calculate TPM for each gene.

Usage

GENEXP_CDS(CDSbygene, flS, Strandtype="NONE")

Arguments

CDSbygene	a genomic ranges of CDS regions for each coding gene
flS	bamfile lists containing the file and path of bam files
Strandtype	strand type of the bam file; "forward" is forwand sequencing, "invert" is reverse sequencing and "NONE" is non-strand specific, Default is "NONE".

Value

The function GENEXP_CDS() return a dataframe containing reads count, TPM for each gene

Author(s)

Ruijia Wang

Examples

## count reads mapped to CDS regions and calculate TPM for each gene
## using forward sequencing
    library("TBX20BamSubset")
    library("Rsamtools")
    library("GenomicAlignments")
    library("GenomicFeatures")
    library("org.Mm.eg.db")
    flsall = getBamFileList()
    extpath = system.file("extdata", "mm9.chr19.refGene.R.DB", package="APAlyzer")
    txdb = loadDb(extpath, packageName='GenomicFeatures')
    IDDB = org.Mm.eg.db
    CDSdbraw = REFCDS(txdb,IDDB)
    DFGENEraw = GENEXP_CDS(CDSdbraw, flsall, Strandtype="forward")

---

PAS2GEF, build reference regions for 3'UTR PASs

Description

Build 3'UTR PAS and IPA (IPA and LE) Reference using GTF file.

Usage

PAS2GEF(GTFfile,AnnoMethod="V2")

Arguments

GTFfile	GTF file of gene annotation
AnnoMethod	annotation method used to build PAS reference, either 'legacy' or 'V2', default is 'V2'

Value

The function PAS2GEF() returns 3 input tables of PAS references: PASREF$refUTRraw is for 3'UTR PAS, PASREF$dfIPA and PASREF$dfLE are for IPA references.

Author(s)

Ruijia Wang

Examples

## build Reference ranges for 3'UTR PASs in mouse
	download.file(url='ftp://ftp.ensembl.org/pub/release-99/gtf/mus_musculus/Mus_musculus.GRCm38.99.gtf.gz',
              destfile='Mus_musculus.GRCm38.99.gtf.gz')			  
	GTFfile="Mus_musculus.GRCm38.99.gtf.gz"	

	PASREF=PAS2GEF(GTFfile, AnnoMethod="V2")	
	refUTRraw=PASREF$refUTRraw
    dfIPA=PASREF$dfIPA
	dfLE=PASREF$dfLE

---

PASEXP_3UTR, calculate relative expression of aUTR and cUTR regions

Description

Map reads to 3'UTR APA regions and calculate relative expression of aUTR and cUTR regions.

Usage

PASEXP_3UTR(UTRdb, flS, Strandtype="NONE")

Arguments

UTRdb	a genomic ranges of aUTR(pPAS to dPAS) and cUTR(cdsend to pPAS) regions for each gene
flS	bamfile lists containing the file and path of bam files
Strandtype	strand type of the bam file; "forward" is forwand sequencing, "invert" is reverse sequencing and "NONE" is non-strand specific, Default is "NONE".

Value

The function PASEXP_3UTR() return a dataframe containning reads count, RPKM and relative expression of aUTR and cUTR for each gene

Author(s)

Ruijia Wang

Examples

## count reads mapped to 3'UTR APA regions and
## calculate relative expression of aUTR and cUTR regions
## using forward sequencing
    library("TBX20BamSubset")
    library("Rsamtools")
    library("GenomicAlignments")
	library("repmis")
    flsall = getBamFileList()
	URL="https://github.com/RJWANGbioinfo/PAS_reference_RData/blob/master/"
	file="mm9_REF.RData"
	source_data(paste0(URL,file,"?raw=True"))
    refUTRraw = refUTRraw[which(refUTRraw$Chrom=='chr19'),]
    UTRdbraw = REF3UTR(refUTRraw)
    DFUTRraw = PASEXP_3UTR(UTRdbraw, flsall, Strandtype="forward")

---

PASEXP_IPA, calculate relative expression of IPA regions

Description

Map reads to IPA regions and calculte relative expression of aUTR and cUTR regions.

Usage

PASEXP_IPA(dfIPAraw, dfLEraw, flS, Strandtype="NONE", nts=1, minMQS=0, SeqType = "SingleEnd")

Arguments

dfIPAraw	a dataframe containing 8 colmuns for Intronic PASs: 'PASid', 'gene_symbol', 'Chrom', 'Strand', 'Pos', 'upstreamSS', 'downstreamSS'. 'upstreamSS' means closest 5'/3' splice site to IPA, 'downstreamSS' means closest 3' splice site
dfLEraw	a dataframe containing 5 colmuns for 3' least exon: 'gene_symbol', 'Chrom', 'Strand', 'LEstart', 'TES'. 'LEstart' means the start position of last 3' exon.
flS	bamfile lists containing the file and path of bam files
Strandtype	strand type of the bam file; "forward" is forwand sequencing, "invert" is reverse sequencing and "NONE" is non-strand specific, Default is "NONE".
nts	number of threads used for computing, parameter used by featureCounts, nthread option, Default is 1
minMQS	minimum mapping quality score of counted reads, parameter used by featureCounts, minMQS option, Default is 0
SeqType	set the sequencing type of reads in bam files can be either 'SingleEnd' (default) or 'ThreeMostPairEnd'.

Value

The function PASEXP_IPA() return a dataframe containning reads count, RPKM and relative expression of aUTR and cUTR for each gene

Author(s)

Ruijia Wang

Examples

## count reads mapped to IPA regions and
## calculte relative expression of aUTR and cUTR regions
## using forward sequencing
    library("TBX20BamSubset")
    library("Rsamtools")
    library("GenomicAlignments")
	library("repmis")
    flsall = getBamFileList()
	URL="https://github.com/RJWANGbioinfo/PAS_reference_RData/blob/master/"
	file="mm9_REF.RData"
	source_data(paste0(URL,file,"?raw=True"))
    IPA_OUTraw=PASEXP_IPA(dfIPA, dfLE, flsall, Strandtype="forward", nts=1)

---

REF3UTR, build reference regions for 3'UTR PASs

Description

Build 3'UTR PAS Reference for distal and proximal PAS.

Usage

REF3UTR(refUTR)

Arguments

refUTR

a dataframe containing 6 colmuns for 3'UTR PASs: 'gene_symbol', 'Chrom', 'Strand', 'Proximal', 'Distal', 'cdsend'

Value

The function REF3UTR() returns a genomic ranges of aUTR(pPAS to dPAS) and cUTR(cdsend to pPAS) regions for each gene

Author(s)

Ruijia Wang

Examples

## build Reference ranges for 3'UTR PASs in mouse
	library(repmis)
	URL="https://github.com/RJWANGbioinfo/PAS_reference_RData/blob/master/"
	file="mm9_REF.RData"
	source_data(paste0(URL,file,"?raw=True"))
    refUTRraw=refUTRraw[which(refUTRraw$Chrom=='chr19'),]
    UTRdbraw=REF3UTR(refUTRraw)

---

REF4PAS, build reference regions for 3'UTR and Intronic PAS using dataframe formated input

Description

build reference regions for 3'UTR and Intronic PAS using dataframe formated input

Usage

REF4PAS(refUTRraw, dfIPAraw, dfLEraw)

Arguments

refUTRraw	a dataframe containing 6 colmuns for 3'UTR PASs: 'gene_symbol', 'Chrom', 'Strand', 'Proximal', 'Distal', 'cdsend'
dfIPAraw	a dataframe containing 8 colmuns for Intronic PASs: 'PASid', 'gene_symbol', 'Chrom', 'Strand', 'Pos', 'upstreamSS', 'downstreamSS'. 'upstreamSS' means closest 5'/3' splice site to IPA, 'downstreamSS' means closest 3' splice site
dfLEraw	a dataframe containing 5 colmuns for 3' least exon: 'gene_symbol', 'Chrom', 'Strand', 'LEstart', 'TES'. 'LEstart' means the start position of last 3' exon.

Value

The function REF4PAS() returns list a genomic ranges of 3'UTR, Intronic PAS and last 3'exon regions for each gene

Author(s)

Ruijia Wang

Examples

## build Reference ranges for 3'UTR and Intronic PAS in mouse (mm9)
	library(repmis)
	URL="https://github.com/RJWANGbioinfo/PAS_reference_RData/blob/master/"
	file="mm9_REF.RData"
	source_data(paste0(URL,file,"?raw=True"))
    refUTRraw=refUTRraw[which(refUTRraw$Chrom=='chr19'),]
	dfIPAraw=dfIPA[which(dfIPA$Chrom=='chr19'),]
	dfLEraw=dfLE[which(dfLE$Chrom=='chr19'),]	
    PASREF=REF4PAS(refUTRraw,dfIPAraw,dfLEraw)
	UTRdbraw=PASREF$UTRdbraw
    dfIPA=PASREF$dfIPA
	dfLE=PASREF$dfLE

---

REFCDS, build reference regions for CDS of protein coding genes

Description

Build CDS reference for protein coding genes.

Usage

REFCDS(txdb,IDDB)

Arguments

txdb	a TranscriptDb generate using GenomicFeatures
IDDB	Genome annotation of the corresponding species, e.g., "org.Hs.eg.db"

Value

The function REFCDS() returns a genomic ranges of CDS regions for each coding gene

Author(s)

Ruijia Wang

Examples

## build Reference ranges for CDS in mouse coding genes
    library("GenomicFeatures")
    library("org.Mm.eg.db")
    extpath = system.file("extdata", "mm9.chr19.refGene.R.DB", package="APAlyzer")
    txdb = loadDb(extpath, packageName='GenomicFeatures')
    IDDB = org.Mm.eg.db
    CDSdbraw = REFCDS(txdb,IDDB)

---

ThreeMostPairBam, extract 3 prime most alignment of a paired-end bam file

Description

extract 3 prime most alignment of a paired-end bam file and saved into a new bam file.

Usage

ThreeMostPairBam(BamfilePath, OutDirPath, StrandType="NONE")

Arguments

BamfilePath	file path of a bam file
OutDirPath	output folder path
StrandType	strand type of the bam file; "forward-reverse": read 1 forward but read 2 is reverse sequencing, "reverse-forward": read 2 forward but read 1 is reverse sequencing, and "NONE" is non-strand specific, Default is "NONE".

Value

The function ThreeMostPairBam() return a single-end bam file containning 3 prime most alignment of the input paired-end file

Author(s)

Ruijia Wang

Examples

## Extract 3 prime most alignment of a paired-end 
## bam file and saved into a new bam file
    library("pasillaBamSubset")
	
    ThreeMostPairBam (BamfilePath=untreated3_chr4(), 
						OutDirPath=getwd(), 
						StrandType='forward-reverse')

---