Workflow: Merge sequence read by fixed length

Load R libraries

library("HaplotypR")
library("ShortRead")

Load example data

Copy example files to a working directory ‘outputDir’:

# Define output directory 
outputDir <- "~/exampleHaplotypR"  
# Create output directoy
if(!dir.exists(outputDir))
  dir.create(outputDir, recursive=T)
  
# Set working directory to output directory
setwd(outputDir)

# Copy example files to output directory
file.copy(from=system.file(package="HaplotypR", "extdata"), to=outputDir, recursive = T)

# List files example files in output direcoty
dir(file.path(outputDir, "extdata"))

The following files should be listed with the last R command: “barcode_Fwd.fasta”, “barcode_Rev.fasta”, “markerFile.txt”, “readsF.fastq.gz”, “readsR.fastq.gz”, “sampleFile.txt”.

Demulitplex sequence reads by sample

Run demultiplexing by sample and rename output files

# set input file path
primerFile <- "extdata/markerFile.txt"
sampleFile <- "extdata/sampleFile.txt"
fnBarcodeF <- "extdata/barcode_Fwd.fasta"
fnBarcodeR <- "extdata/barcode_Rev.fasta"
reads <- list.files("extdata", pattern="reads", full.names = T)

# create output subdirectory 
outDeplexSample <- file.path(outputDir, "dePlexSample")
dir.create(outDeplexSample)

# demultiplex by samples
dePlexSample <- demultiplexReads(reads[1], reads[2], fnBarcodeF, fnBarcodeR, outDeplexSample)

# rename output files to sample files
sampleTab <- read.delim(sampleFile, stringsAsFactors=F)
dePlexSample <- renameDemultiplexedFiles(sampleTab, dePlexSample)

# save summary table
write.table(dePlexSample, file.path(outputDir, "demultiplexSampleSummary.txt"), sep="\t", row.names=F)

Demulitplex sequence reads by marker

Run demultiplex by marker and truncate primer sequence

# create output subdirectory 
outDeplexMarker <- file.path(outputDir, "dePlexMarker")
dir.create(outDeplexMarker)
  
# process each marker
markerTab <- read.delim(primerFile, stringsAsFactors=F)
dePlexMarker <- demultiplexByMarker(dePlexSample, markerTab, outDeplexMarker)

# save summary table
write.table(dePlexMarker, file.path(outputDir, "demultiplexMarkerSummary.txt"), sep="\t", row.names=F)

Merge paired sequence read by fixed length

Fuse paired reads. Two methods are provided for fusing paired reads. First method works for non overlapping sequence read pairs. Trim to a fixed length (removes low quality bases) and then concatenate forward and reverse read. Second method work only for overlapping sequence read pair by merging the overlap of the forward and reverse read (using vsearch wrapper).

# create output subdirectory 
outProcFiles <- file.path(outputDir, "processedReads")
dir.create(outProcFiles)

# Trim options
numNtF <- 190
numNtR <- 120
postfix <- sprintf("_bind%.0f_%.0f", numNtF, numNtR)

# Adjust reference to trim options and save as fasta file
refSeq <- as.character(markerTab$ReferenceSequence)
refSeq <- DNAStringSet(paste(substr(refSeq, 1,numNtF), substr(refSeq, nchar(refSeq)+1-numNtR, nchar(refSeq)), sep=""))
names(refSeq) <- markerTab$MarkerID
lapply(seq_along(refSeq), function(i){
  writeFasta(refSeq[i], file.path(outputDir, paste(names(refSeq)[i], postfix, ".fasta", sep="")))
})

# Fuse paired read
procReads <- bindAmpliconReads(as.character(dePlexMarker$FileR1), as.character(dePlexMarker$FileR2), outProcFiles, 
                         read1Length=numNtF, read2Length=numNtR)
procReads <- cbind(dePlexMarker[,c("SampleID", "SampleName","BarcodePair", "MarkerID")], procReads)
write.table(procReads, file.path(outputDir, sprintf("processedReadSummary%s.txt", postfix)), sep="\t", row.names=F)

Call SNPs

Calculate mismatch rate and call SNPs

# Options
minMMrate <- 0.5
minOccGen <- 2

# process each marker
snpLst <- lapply(markerTab$MarkerID, function(marker){
  # Calculate mismatch rate
  seqErrLst <- calculateMismatchFrequencies(as.character(procReads[procReads$MarkerID == marker, "ReadFile"]), 
                                            refSeq[marker], 
                                            method ="pairwiseAlignment", # c("pairwiseAlignment","compareDNAString"), 
                                            minCoverage=100L)
  names(seqErrLst) <- procReads[procReads$MarkerID == marker, "SampleID"]
  seqErr <- do.call(cbind, lapply(seqErrLst, function(l){
    l[,"MisMatch"]/l[,"Coverage"]
  }))
  write.table(seqErr, file.path(outputDir, sprintf("mismatchRate_rate_%s%s.txt", marker, postfix)), sep="\t", row.names=F)
  
  # Call SNPs
  potSNP <- callGenotype(seqErr, minMismatchRate=minMMrate, minReplicate=minOccGen)
  snpRef <- unlist(lapply(potSNP, function(snp){
    as.character(subseq(refSeq[marker], start=snp, width=1))
  }))
  snps <- data.frame(Chr=marker, Pos=potSNP, Ref=snpRef, Alt="N", stringsAsFactors=F)
  write.table(snps, file=file.path(outputDir, sprintf("potentialSNPlist_rate%.0f_occ%i_%s%s.txt", 
                                                  minMMrate*100, minOccGen, marker, postfix)), 
              row.names=F, col.names=T, sep="\t", quote=F)
  
  # Plot mismatch rate and SNP calls
  png(file.path(outputDir, sprintf("plotMisMatchRatePerBase_rate%.0f_occ%i_%s%s.png", 
                             minMMrate*100, minOccGen, marker, postfix)), 
      width=1500 , height=600)
  matplot(seqErr, type="p", pch=16, cex=0.4, col="#00000088", ylim=c(0, 1),
          ylab="Mismatch Rate", xlab="Base Position", main=marker, cex.axis=2, cex.lab=2)
  abline(v=snps[,"Pos"], lty=2, col="grey")
  abline(h=minMMrate, lty=1, col="red")
  dev.off()
  
  return(snps)
})
names(snpLst) <- markerTab$MarkerID

Call Haplotypes

# call haplotype options
minCov <- 3
detectionLimit <- 1/100
minOccHap <- 2
minCovSample <- 25

# call final haplotypes
finalTab <- createFinalHaplotypTable(
  outputDir = outputDir, sampleTable = procReads, markerTable = markerTab, referenceSeq = refSeq,
  snpList = snpLst, postfix = postfix, minHaplotypCoverage = minCov, minReplicate = minOccHap, 
  detectability = detectionLimit, minSampleCoverage = minCovSample)

Get haplotyp list and sequence

Todo