gacatag: June 2019

Ali Oghabian

2019-04-25

The topics covered in this post are:

R and IntEREst version
Files in the zip
Annotating u12 type introns of HG38
ncRNAs with U12-type introns
U12 annotation comparison
Reference

Files in the zip

You can download the zip file the includes all the scripts and R objects from here. The files in the zip/folder include:

annoFil.rda R object file. A data frame that includes various annotations and information for the latest ENSEMBL genes (GRCh38.p12).
AllU12Introns.tsv Tab separated text file. Includes the detecetd U12-type introns and their annotations.
ens38UncolMod.rda ENSEMBL reference that includes gene and transcript IDs together with coordinates of the introns and exons.
ensHg38UncolFilpaste60AnnoMat_40_don07_bp14.rda R object file. Data frame with U12/U2 type annotation of the reference (hg38) introns, the U12 GT-AG or AT-AC subtype intfo and the PWM match scores.
allIntronsU12annotationGeneType.tsv Tab separated text file. Includes all annotated U12 type introns.
nonProtCodingU12Introns.tsv Tab separated text file. Includes the detecetd U12 type introns that were annotated as NOT protein-coding.
report.Rmd an RMarkdown file that was used to generate this report.
report.html the HTML file that was generated from report.Rmd.
U12LncRNA.tsv Tab separated text file. Includes the detecetd U12-type introns that were annotated to be lncRNAs.

R and IntEREst version

Please note the R version and the IntEREst version used for these analysis:

R.Version()

## $platform
## [1] "x86_64-pc-linux-gnu"
## 
## $arch
## [1] "x86_64"
## 
## $os
## [1] "linux-gnu"
## 
## $system
## [1] "x86_64, linux-gnu"
## 
## $status
## [1] "Under development (unstable)"
## 
## $major
## [1] "3"
## 
## $minor
## [1] "6.0"
## 
## $year
## [1] "2018"
## 
## $month
## [1] "05"
## 
## $day
## [1] "02"
## 
## $`svn rev`
## [1] "74682"
## 
## $language
## [1] "R"
## 
## $version.string
## [1] "R Under development (unstable) (2018-05-02 r74682)"
## 
## $nickname
## [1] "Unsuffered Consequences"

packageVersion("IntEREst")

## [1] '1.7.5'

Annotating u12 type introns of HG38

Initially, a reference data frame was produced from ENSEMBL HG38 (GRCh38.p12). Note that in order to be able to run the R scripts in the document you should set the R working directory to the folder that contains the extrcated files form the zip file.

# Time demanding
ens38Uncol<- referencePrepare (sourceBuild="biomaRt", 
    biomart="ENSEMBL_MART_ENSEMBL",
    biomartDataset="hsapiens_gene_ensembl",
    biomartHost="www.ensembl.org", 
    ignore.strand=FALSE, 
    annotateGeneIds=TRUE, 
    collapseExons=FALSE)

Next the U12-type introns were annotated using donor socre threoshold 0.07 and branch point score threshold 0.14. If an intron was not annotated as a U12-type or a U2-type we considered it as U2-type (i.e. the default parameter setting setNaAs= "U2" was used).

# Time demanding
library(BSgenome.Hsapiens.UCSC.hg38)
# Building PWM
pwmPaste<-buildSsTypePwms(u12DonorBegin=13, u2DonorBegin=11, 
    u2AcceptorBegin=38, u12DonorEnd=17, u2DonorEnd=17, 
    u2AcceptorEnd=40, pasteSites=TRUE)
save(pwmPaste, file="./pwmPaste.rda")

# Ignore 3' end nucleotide
# Annotations will be based on Donor and BP scores
accPasteScore=as.matrix(pwmPaste[[3]])
for(i in 1:nrow(accPasteScore))accPasteScore[i,1]=0

# Modify the chr names of the reference data.frame

if(length(grep("^chr",ens38Uncol$chr))==0)
    ens38Uncol$chr<- paste("chr", ens38Uncol$chr, sep="")

accPasteScore=as.matrix(pwmPaste[[3]])
for(i in 1:nrow(accPasteScore))accPasteScore[i,1]=0

ens38UncolMod<- ens38Uncol[which(ens38Uncol$chr %in% 
    paste("chr", c(1:22,"X","Y", "MT"), sep="")),]
ens38UncolMod[which(ens38UncolMod$chr == "chrMT"),]<- "chrM"
save(ens38UncolMod, file="./ens38UncolMod.rda")

# Annotate U12-type introns using donor and BP of U12- and Donor and acceptor 
# sites of U2-type introns.

ensHg38UncolFilpaste60AnnoMat_40_don07_bp14<- annotateU12(
    pwmU12U2= list(
        pwmPaste[[1]],
        pwmPaste[[2]],
        accPasteScore,
        pwmPaste[[4]],
        pwmPaste[[5]]),
    pwmSsIndex= list(
        indexDonU12=1, 
        indexBpU12=1, 
        indexAccU12=1, 
        indexDonU2=1, 
        indexAccU2=1), 
    referenceChr= ens38UncolMod[,"chr"], 
    referenceBegin= as.numeric(ens38UncolMod[,"begin"]), 
    referenceEnd= as.numeric(ens38UncolMod[,"end"]), 
    referenceIntronExon= ens38UncolMod[,"int_ex"],
    intronExon= "intron",
    matchWindowRelativeUpstreamPos= c(2,-40,NA,NA,NA),
    matchWindowRelativeDownstreamPos= c(6,-3,NA,NA,NA), 
    minMatchScore= c( .07, .14, 0, .07,  0), 
    refGenome= BSgenome.Hsapiens.UCSC.hg38, 
    setNaAs= "U2", 
    annotateU12Subtype= TRUE,
    includeMatchScores= TRUE)

save(ensHg38UncolFilpaste60AnnoMat_40_don07_bp14, 
    file="./ensHg38UncolFilpaste60AnnoMat_40_don07_bp14.rda")

The number of the detected U12-type introns are as follows:

load(file="./ensHg38UncolFilpaste60AnnoMat_40_don07_bp14.rda")

# Number of U12-type intron
table(ensHg38UncolFilpaste60AnnoMat_40_don07_bp14$int_type)

## 
##     U12      U2 
##    3528 1051845

# Number of unique U12-type intron
indU12<- which(ensHg38UncolFilpaste60AnnoMat_40_don07_bp14$int_type=="U12")
length(
    unique(paste(ens38UncolMod[indU12,"chr"], 
        ens38UncolMod[indU12,"begin"], ens38UncolMod[indU12,"end"], sep="_")))

## [1] 826

# Number of U12-type intron subtypes
table(ensHg38UncolFilpaste60AnnoMat_40_don07_bp14$u12_subtype)

## 
## AT-AC GT-AG 
##   903  2625

# Number of unique U12-type intron subtypes
uniCoord<- unique(paste(ens38UncolMod[indU12,"chr"], 
        ens38UncolMod[indU12,"begin"], ens38UncolMod[indU12,"end"], sep="_"))
coord<- paste(ens38UncolMod[indU12,"chr"], 
        ens38UncolMod[indU12,"begin"], ens38UncolMod[indU12,"end"], sep="_")
indUni<-match(uniCoord, coord)
table(ensHg38UncolFilpaste60AnnoMat_40_don07_bp14$u12_subtype[indU12[indUni]])

## 
## AT-AC GT-AG 
##   195   631

ncRNAs with U12-type introns

To detect the ncRNAs, initially various annotations of ENSEMBL genes, e.g. extrenal gene name and biotype (description), were downloaded using Using biomaRt.

ensembl<- biomaRt::useMart("ENSEMBL_MART_ENSEMBL", 
    host="www.ensembl.org", dataset="hsapiens_gene_ensembl")

annoFil<- biomaRt::getBM( attributes=c("ensembl_gene_id", 
        "ensembl_transcript_id", "external_gene_name", "gene_biotype", 
        "transcript_biotype"), 
    filters = "ensembl_transcript_id", 
    values = unique(ens38UncolMod$transcript_id), 
    mart=ensembl, uniqueRows = TRUE)

save(annoFil, file="./annoFil.rda")


indPick<- which((transcript_biotype=="lincRNA" | 
    transcript_biotype=="bidirectional_promoter_lncRNA") & 
    ensHg38UncolFilpaste60AnnoMat_40_don07_bp14$int_type=="U12")
U12Linc<-ens38UncolMod[indPick,]

donPos=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=U12Linc[U12Linc[,4]=="+",1],
                    start=
                        as.numeric(U12Linc[U12Linc[,4]=="+",2]),
                    end=
                        as.numeric(U12Linc[U12Linc[,4]=="+",2])+9,
                    as.character=TRUE)
accNeg=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=U12Linc[U12Linc[,4]=="-",1],
                    start=
                        as.numeric(U12Linc[U12Linc[,4]=="-",2]),
                    end=
                        as.numeric(U12Linc[U12Linc[,4]=="-",2])+39,
                    as.character=TRUE)

accPos=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=U12Linc[U12Linc[,4]=="+",1],
                    start=as.numeric(U12Linc[U12Linc[,4]=="+",3])-39,
                    end=as.numeric(U12Linc[U12Linc[,4]=="+",3])
                        ,
                    as.character=TRUE)
donNeg=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=U12Linc[U12Linc[,4]=="-",1],
                    start=as.numeric(U12Linc[U12Linc[,4]=="-",3])-9,
                    end=as.numeric(U12Linc[U12Linc[,4]=="-",3]),
                    as.character=TRUE)

donNeg<- as.vector(
    as.character(Biostrings::reverseComplement(DNAStringSet(donNeg))))
accNeg<- as.vector(
    as.character(Biostrings::reverseComplement(DNAStringSet(accNeg))))
accPos<- as.vector(accPos)
donPos<- as.vector(donPos)

donorSeq<- rep("",nrow(U12Linc))
acceptorSeq<- rep("",nrow(U12Linc))
donorSeq[U12Linc[,4]=="+"]<-donPos
donorSeq[U12Linc[,4]=="-"]<-donNeg
acceptorSeq[U12Linc[,4]=="+"]<-accPos
acceptorSeq[U12Linc[,4]=="-"]<-accNeg
U12LincMat<- cbind(U12Linc, donorSeq, acceptorSeq)

# Write lincRNA U12-type introns 
write.table(U12LincMat, file="./U12LncRNA.tsv", 
    col.names=T, row.names=F, sep='\t', quote=F)
# write data frame with info for all introns
write.table(
    cbind(ens38UncolMod, external_gene_name, 
        ensHg38UncolFilpaste60AnnoMat_40_don07_bp14, 
        transcript_biotype),
    file="./allIntronsU12annotationGeneType.tsv",
    col.names=T, row.names=F, sep='\t', quote=F)

# Build datat frame with U12 type introns info
u12Out<- 
    cbind(ens38UncolMod, ensHg38UncolFilpaste60AnnoMat_40_don07_bp14,
            external_gene_name, 
            transcript_biotype)[which(
                ensHg38UncolFilpaste60AnnoMat_40_don07_bp14$int_type=="U12"),]

donPos=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=u12Out[u12Out[,4]=="+",1],
                    start=
                        as.numeric(u12Out[u12Out[,4]=="+",2]),
                    end=
                        as.numeric(u12Out[u12Out[,4]=="+",2])+9,
                    as.character=TRUE)
accNeg=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=u12Out[u12Out[,4]=="-",1],
                    start=
                        as.numeric(u12Out[u12Out[,4]=="-",2]),
                    end=
                        as.numeric(u12Out[u12Out[,4]=="-",2])+39,
                    as.character=TRUE)

accPos=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=u12Out[u12Out[,4]=="+",1],
                    start=as.numeric(u12Out[u12Out[,4]=="+",3])-39,
                    end=as.numeric(u12Out[u12Out[,4]=="+",3])
                        ,
                    as.character=TRUE)
donNeg=Biostrings::getSeq(BSgenome.Hsapiens.UCSC.hg38, 
                    names=u12Out[u12Out[,4]=="-",1],
                    start=as.numeric(u12Out[u12Out[,4]=="-",3])-9,
                    end=as.numeric(u12Out[u12Out[,4]=="-",3]),
                    as.character=TRUE)

donNeg<- 
as.vector(as.character(Biostrings::reverseComplement(DNAStringSet(donNeg))))
accNeg<- 
as.vector(as.character(Biostrings::reverseComplement(DNAStringSet(accNeg))))
accPos<- as.vector(accPos)
donPos<- as.vector(donPos)

donorSeq<- rep("",nrow(u12Out))
acceptorSeq<- rep("",nrow(u12Out))
donorSeq[u12Out[,4]=="+"]<-donPos
donorSeq[u12Out[,4]=="-"]<-donNeg
acceptorSeq[u12Out[,4]=="+"]<-accPos
acceptorSeq[u12Out[,4]=="-"]<-accNeg
u12OutSeq<- cbind(u12Out, donorSeq, acceptorSeq)

write.table(
    u12OutSeq,
    file="./AllU12Introns.tsv",
    col.names=T, row.names=F, sep='\t', quote=F)
write.table(
    u12OutSeq[which(u12OutSeq$transcript_biotype!="protein_coding"),],
    file="./nonProtCodingU12Introns.tsv",
    col.names=T, row.names=F, sep='\t', quote=F)

U12 annotation comparison

Changes in the parameter settings of the annotateU12() function may lead to detection of different sets of U12-type introns. Here, we compare the U12-type introns detected from the same annotateU12() run as mentioned above (on a reference built from HG19) to those reported by Merico, D. et al.

Detected U12 type introns for HG19 compared to those detected by merico, D. et al.

Finally

The following scripts were used in R to generate the suppl1.html file from the suppl1.Rmd file.

library("rmarkdown")
render("./report.Rmd")

Reference

Merico,D. et al. (2015) Compound heterozygous mutations in the noncoding RNU4ATAC cause Roifman Syndrome by disrupting minor intron splicing. Nat Commun, 6, 8718.

gacatag

Detecting U12-type introns using IntEREst R/Biocondcutor package