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| #' call primer3 for a given set of DNAstringSet object | |
| #' | |
| #' @param seq DNAstring object, one DNA string for the given amplicon | |
| #' @param size_range default: '151-500' | |
| #' @param Tm melting temprature parameters default:c(55,57,58) | |
| #' @param name name of the amplicon in chr_start_end format | |
| #' @param primer3 primer3 location | |
| #' @param therme.param thermodynamic parameters folder | |
| #' @param settings text file for parameters | |
| #' @author Altuna Akalin modified Arnaud Krebs' original function | |
| #' @example | |
| #' | |
| #' primers=mapply(.callP3NreadOrg,seq=sample.seq, | |
| #' name=names(sample.seq), | |
| #' MoreArgs=list(primer3=primer3,size_range=size_range,Tm=Tm, | |
| #' thermo.param=thermoParam,settings=settings) | |
| #' ,SIMPLIFY = FALSE) | |
| #' | |
| #' | |
| .callP3NreadOrg<-function(seq,size_range='151-500',Tm=c(55,57,58),name, | |
| primer3="/work2/gschub/arnaud/softwares/primer3/primer3-2.3.4/bin/primer3_core", | |
| thermo.param="/work2/gschub/arnaud/softwares/primer3/primer3-2.3.4/src/primer3_config/", | |
| settings="/work2/gschub/arnaud/softwares/primer3/primer3-2.3.4/default_settings.txt"){ | |
| #print(excluded.regions) | |
| # make primer 3 input file | |
| p3.input=tempfile() | |
| p3.output=tempfile() | |
| write(paste(sprintf("SEQUENCE_ID=%s\n",name), | |
| sprintf("SEQUENCE_TEMPLATE=%s\n",as.character(seq)), | |
| "PRIMER_TASK=pick_detection_primers\n", | |
| "PRIMER_PICK_LEFT_PRIMER=1\n" , | |
| "PRIMER_PICK_INTERNAL_OLIGO=0\n", | |
| "PRIMER_PICK_RIGHT_PRIMER=1\n", | |
| "PRIMER_EXPLAIN_FLAG=1\n", | |
| "PRIMER_PAIR_MAX_DIFF_TM=3\n", | |
| sprintf("PRIMER_MIN_TM=%s\n" ,Tm[1]), | |
| sprintf("PRIMER_OPT_TM=%s\n" ,Tm[2]), | |
| sprintf("PRIMER_MAX_TM=%s\n" ,Tm[3]), | |
| sprintf("PRIMER_PRODUCT_SIZE_RANGE=%s\n" ,size_range), | |
| sprintf("PRIMER_THERMODYNAMIC_PARAMETERS_PATH=%s\n" ,thermo.param), | |
| "=", | |
| sep=''), | |
| p3.input) | |
| #call primer 3 and store the output in a temporary file | |
| try(system( | |
| paste(primer3 ,p3.input, "-p3_settings_file",settings, | |
| ">", p3.output) | |
| )) | |
| #import and parse the output into a dataframe named designed.primers | |
| out=read.delim(p3.output, sep='=', header=FALSE) | |
| unlink(c(p3.input,p3.output) ) # delete temp files | |
| returned.primers=as.numeric(as.vector(out[out[,1]=='PRIMER_PAIR_NUM_RETURNED',][,2])) | |
| if (length(returned.primers)==0){ warning('primers not detected for ',name,call. = FALSE);return(NA)} | |
| if ((returned.primers)==0){ warning('primers not detected for ',name,call. = FALSE);return(NA)} | |
| if (returned.primers>0){ | |
| designed.primers=data.frame() | |
| for (i in seq(0,returned.primers-1,1)){ | |
| #IMPORT SEQUENCES | |
| id=sprintf( 'PRIMER_LEFT_%i_SEQUENCE',i) | |
| PRIMER_LEFT_SEQUENCE=as.character(out[out[,1]==id,][,2]) | |
| id=sprintf( 'PRIMER_RIGHT_%i_SEQUENCE',i) | |
| PRIMER_RIGHT_SEQUENCE=as.character(out[out[,1]==id,][,2]) | |
| #IMPORT PRIMING POSITIONS | |
| id=sprintf( 'PRIMER_LEFT_%i',i) | |
| PRIMER_LEFT=as.numeric(unlist(strsplit(as.vector((out[out[,1]==id,][,2])),','))) | |
| #PRIMER_LEFT_LEN=as.numeric(unlist(strsplit(as.vector((out[out[,1]==id,][,2])),','))) | |
| id=sprintf( 'PRIMER_RIGHT_%i',i) | |
| PRIMER_RIGHT=as.numeric(unlist(strsplit(as.vector((out[out[,1]==id,][,2])),','))) | |
| #IMPORT Tm | |
| id=sprintf( 'PRIMER_LEFT_%i_TM',i) | |
| PRIMER_LEFT_TM=as.numeric(as.vector((out[out[,1]==id,][,2])),',') | |
| id=sprintf( 'PRIMER_RIGHT_%i_TM',i) | |
| PRIMER_RIGHT_TM=as.numeric(as.vector((out[out[,1]==id,][,2])),',') | |
| res=out[grep(i,out[,1]),] | |
| extra.inf=t(res)[2,,drop=FALSE] | |
| colnames(extra.inf)=sub( paste("_",i,sep=""),"",res[,1]) | |
| extra.inf=extra.inf[,-c(4:9),drop=FALSE] # remove redundant columns | |
| extra.inf=apply(extra.inf,2,as.numeric) | |
| #Aggegate in a dataframe | |
| primer.info=data.frame(i, | |
| PRIMER_LEFT_SEQUENCE,PRIMER_RIGHT_SEQUENCE, | |
| PRIMER_LEFT_TM, PRIMER_RIGHT_TM, | |
| PRIMER_LEFT_pos=PRIMER_LEFT[1], | |
| PRIMER_LEFT_len=PRIMER_LEFT[2], | |
| PRIMER_RIGHT_pos=PRIMER_RIGHT[1], | |
| PRIMER_RIGHT_len=PRIMER_RIGHT[2], | |
| t(data.frame(extra.inf)) | |
| ) | |
| rownames(primer.info)=NULL | |
| designed.primers=rbind(designed.primers, primer.info) | |
| #print(primer.info) | |
| } | |
| #colnames(designed.primers)=c('PrimerID', | |
| # 'Fwseq','Rvseq', | |
| # 'FwTm','RvTm', | |
| # 'FwPos','Fwlen', | |
| # 'RvPos','Rvlen', | |
| # 'fragLen' ) | |
| } | |
| return(designed.primers) | |
| } | |
| #' convert primer list to genomic intervals | |
| #' | |
| #' function returns a GRanges or data frame object from a list of primers designed | |
| #' by \code{designPrimers} function after calculating genomic location of the amplicon | |
| #' targeted by the primers. | |
| #' | |
| #' @param primers a list of primers returned by \code{designPrimers} function | |
| #' @param as.data.frame logical indicating if a data frame should be returned | |
| #' instead of \code{GRanges} object. | |
| #' | |
| #' @examples | |
| #' data(bisPrimers) | |
| #' # remove data with no primers found | |
| #' bisPrimers=bisPrimers[!is.na(bisPrimers)] | |
| #' gr.pr=primers2ranges(bisPrimers) # convert primers to GRanges | |
| #' | |
| #' @seealso \code{\link{filterPrimers}}, \code{\link{designPrimers}} | |
| #' | |
| #' @export | |
| #' @docType methods | |
| primers2ranges<-function(primers,as.data.frame=FALSE){ | |
| if(any(is.na(primers))){ | |
| warning( "There are targets without primers\nfiltering those before conversion") | |
| primers=primers[ !is.na(primers) ] | |
| } | |
| df=do.call("rbind",primers) # get primers to a df | |
| locs=gsub("\\|\\.+","",rownames(df)) # get the coordinates from list ids | |
| temp=do.call("rbind",strsplit(locs,"_")) # | |
| start=as.numeric(temp[,2]) | |
| chr=as.character(temp[,1]) | |
| amp.start= start + as.numeric(df$PRIMER_LEFT_pos) | |
| amp.end = start + as.numeric(df$PRIMER_RIGHT_pos) | |
| res=data.frame(chr=chr,start=amp.start,end=amp.end,df) | |
| #saveRDS(res,file="/work2/gschub/altuna/projects/DMR_alignments/all.designed.primers.to.amps.rds") | |
| if(as.data.frame) | |
| { | |
| return(res) | |
| } | |
| gr=GRanges(seqnames=res[,1],ranges=IRanges(res[,2],res[,3]) ) | |
| values(gr)=res[,-c(1,2,3)] | |
| gr | |
| } | |
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