inst/scripts/generateValuesForUnitTestsTrans.R
In fmicompbio/mutscan: Preprocessing and Analysis of Deep Mutational Scanning Data
suppressPackageStartupMessages({
library(Biostrings)
library(ShortRead)
library(dplyr)
})
collapse_seqs <- function(seqs, umis, reads, maxdist, umimaxdist, minreads, minratio) {
tokeep <- list()
while (length(seqs) > 0) {
if (reads[1] >= minreads) {
d <- stringdist::stringdist(seqs[1], seqs, method = "hamming")
w <- union(1, which(d <= maxdist & reads[1] >= minratio * reads))
} else {
w <- 1
}
tokeep[[seqs[1]]] <- paste(umis[w], collapse = ",")
seqs <- seqs[-w]
umis <- umis[-w]
reads <- reads[-w]
}
tokeep <- lapply(tokeep, function(tk) {
tk <- sort(unique(strsplit(tk, ",")[[1]]))
if (length(tk) == 1) {
tk
} else {
k <- c()
while (length(tk) > 0) {
d <- stringdist::stringdist(tk[1], tk, method = "hamming")
w <- which(d <= umimaxdist)
k <- c(k, tk[1])
tk <- tk[-w]
}
k
}
})
tokeep
}
processReadsTrans <- function(Ldef) {
## Read data
fq1 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqForward)
if (!is.null(Ldef$fastqReverse)) {
fq2 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqReverse)
}
## Number of reads
message("Number of reads: ", length(fq1)) ## 1000
## Adapter sequences
adapt <- Biostrings::vcountPattern(Ldef$adapterForward, fq1) > 0
if (!is.null(Ldef$fastqReverse)) {
adapt2 <- Biostrings::vcountPattern(Ldef$adapterReverse, fq2) > 0
adapt <- adapt | adapt2
}
message("Number of reads with adapters: ", sum(adapt)) ## 314
fq1 <- fq1[!adapt]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!adapt]
}
## Average quality in variable sequences too low
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
avgQualForward <- ShortRead::alphabetScore(Biostrings::quality(varForward))/
BiocGenerics::width(varForward)
lowqual <- avgQualForward < Ldef$avePhredMinForward
if (!is.null(Ldef$fastqReverse)) {
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
avgQualReverse <- ShortRead::alphabetScore(Biostrings::quality(varReverse))/
BiocGenerics::width(varReverse)
lowqual <- lowqual | avgQualReverse < Ldef$avePhredMinReverse
}
message("Number of reads with low average quality: ", sum(lowqual)) # 7
fq1 <- fq1[!lowqual]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!lowqual]
}
## Too many Ns in variable sequences
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
nbrNForward <- Biostrings::vcountPattern("N", varForward, fixed = TRUE)
toomanynvar <- nbrNForward > Ldef$variableNMaxForward
if (!is.null(Ldef$fastqReverse)) {
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
nbrNReverse <- Biostrings::vcountPattern("N", varReverse, fixed = TRUE)
toomanynvar <- toomanynvar | nbrNReverse > Ldef$variableNMaxReverse
}
message("Number of reads with too many N in variable sequence: ", sum(toomanynvar)) # 0
fq1 <- fq1[!toomanynvar]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanynvar]
}
## Too many Ns in UMIs
umiForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + 1, width = Ldef$umiLengthForward)
if (!is.null(Ldef$fastqReverse)) {
umiReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + 1, width = Ldef$umiLengthReverse)
umis <- Biostrings::xscat(umiForward, umiReverse)
} else {
umis <- umiForward
}
nbrNumi <- Biostrings::vcountPattern("N", umis, fixed = TRUE)
toomanynumi <- nbrNumi > Ldef$umiNMax
message("Number of reads with too many N in UMI: ", sum(toomanynumi)) # 0
fq1 <- fq1[!toomanynumi]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanynumi]
}
## Compare to wildtype, forward
mutCodons <- NULL
uniqueMutCodons <- NULL
mutBases <- NULL
uniqueMutBases <- NULL
nbrMutBasesTot <- NULL
if (Ldef$wildTypeForward != "") {
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
p <- DNAStringSet(rep(Ldef$wildTypeForward, length(varForward)))
pattern_width <- width(p)
subject_width <- width(varForward)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(varForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
## First check, excluding reads with more than 3x(max nbr mut codons) mismatches
baseMismatches <- mismatchPositions
nbrMutBases <- S4Vectors::elementNROWS(unique(baseMismatches))
toomanybases <- nbrMutBases > 3 * Ldef$nbrMutatedCodonsMaxForward
message("Number of reads with too many mutated codons, forward (1): ", sum(toomanybases))
fq1 <- fq1[!toomanybases]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanybases]
}
mismatchPositions <- mismatchPositions[!toomanybases]
varForward <- varForward[!toomanybases]
mismatchQualities <- Biostrings::quality(varForward)[mismatchPositions]
lowqualmm <- min(as(mismatchQualities, "IntegerList")) < Ldef$mutatedPhredMinForward
message("Number of reads with low-quality mismatch bases, forward: ", sum(lowqualmm)) ## 0
fq1 <- fq1[!lowqualmm]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!lowqualmm]
}
mismatchPositions <- mismatchPositions[!lowqualmm]
varForward <- varForward[!lowqualmm]
## Number of mutated codons
codonMismatches <- (mismatchPositions - 1) %/% 3 + 1
nbrMutCodons <- S4Vectors::elementNROWS(unique(codonMismatches))
toomanycodons <- nbrMutCodons > Ldef$nbrMutatedCodonsMaxForward
message("Number of reads with too many mutated codons, forward: ", sum(toomanycodons)) ## 287
fq1 <- fq1[!toomanycodons]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanycodons]
}
codonMismatches <- codonMismatches[!toomanycodons]
varForward <- varForward[!toomanycodons]
nbrMutCodons <- nbrMutCodons[!toomanycodons]
nbrMutBases <- S4Vectors::elementNROWS(unique(mismatchPositions[!toomanycodons]))
## Forbidden codons
uniqueMutCodons <- unique(codonMismatches)
mutCodons <- S4Vectors::endoapply(uniqueMutCodons,
function(v) rep(3 * v, each = 3) + c(-2, -1, 0))
mutCodons <- as(varForward, "DNAStringSet")[mutCodons]
matchForbidden <- Biostrings::vmatchPattern(
pattern = Ldef$forbiddenMutatedCodonsForward,
as(mutCodons, "DNAStringSet"),
fixed = FALSE)
forbiddencodon <- sapply(BiocGenerics::relist(start(unlist(matchForbidden)) %% 3==1,
matchForbidden), function(i) any(i))
message("Number of reads with forbidden codons, forward: ", sum(forbiddencodon)) ## 6
fq1 <- fq1[!forbiddencodon]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!forbiddencodon]
}
mutCodons <- mutCodons[!forbiddencodon]
nbrMutCodonsTot <- nbrMutCodons[!forbiddencodon]
nbrMutBasesTot <- nbrMutBases[!forbiddencodon]
uniqueMutCodons <- uniqueMutCodons[!forbiddencodon]
splitMutCodons <- as(strsplit(gsub("([^.]{3})", "\\1\\.",
as.character(mutCodons)), ".", fixed = TRUE),
"CharacterList")
encodedMutCodonsForward <- S4Vectors::unstrsplit(BiocGenerics::relist(
paste0("f", unlist(uniqueMutCodons), unlist(splitMutCodons)),
uniqueMutCodons),
sep = "_")
} else {
encodedMutCodonsForward <- ""
}
## Compare to wildtype, reverse
if (!is.null(Ldef$fastqReverse) && Ldef$wildTypeReverse != "") {
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
p <- DNAStringSet(rep(Ldef$wildTypeReverse, length(varReverse)))
pattern_width <- width(p)
subject_width <- width(varReverse)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(varReverse)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
## First check, excluding reads with more than 3x(max nbr mut codons) mismatches
baseMismatches <- mismatchPositions
nbrMutBases <- S4Vectors::elementNROWS(unique(baseMismatches))
toomanybases <- nbrMutBases > 3 * Ldef$nbrMutatedCodonsMaxReverse
message("Number of reads with too many mutated codons, reverse (1): ", sum(toomanybases))
fq1 <- fq1[!toomanybases]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanybases]
}
mismatchPositions <- mismatchPositions[!toomanybases]
varReverse <- varReverse[!toomanybases]
encodedMutCodonsForward <- encodedMutCodonsForward[!toomanybases]
nbrMutBasesTot <- nbrMutBasesTot[!toomanybases]
nbrMutCodonsTot <- nbrMutCodonsTot[!toomanybases]
mismatchQualities <- Biostrings::quality(varReverse)[mismatchPositions]
lowqualmm <- min(as(mismatchQualities, "IntegerList")) < Ldef$mutatedPhredMinReverse
message("Number of reads with low-quality mismatch bases, reverse: ", sum(lowqualmm)) ## 0
fq1 <- fq1[!lowqualmm]
fq2 <- fq2[!lowqualmm]
mismatchPositions <- mismatchPositions[!lowqualmm]
varReverse <- varReverse[!lowqualmm]
encodedMutCodonsForward <- encodedMutCodonsForward[!lowqualmm]
nbrMutBasesTot <- nbrMutBasesTot[!lowqualmm]
nbrMutCodonsTot <- nbrMutCodonsTot[!lowqualmm]
## Number of mutated codons
codonMismatches <- (mismatchPositions - 1) %/% 3 + 1
nbrMutCodons <- S4Vectors::elementNROWS(unique(codonMismatches))
toomanycodons <- nbrMutCodons > Ldef$nbrMutatedCodonsMaxReverse
message("Number of reads with too many mutated codons, reverse: ", sum(toomanycodons)) ## 105
fq1 <- fq1[!toomanycodons]
fq2 <- fq2[!toomanycodons]
codonMismatches <- codonMismatches[!toomanycodons]
varReverse <- varReverse[!toomanycodons]
encodedMutCodonsForward <- encodedMutCodonsForward[!toomanycodons]
nbrMutBasesTot <- nbrMutBasesTot[!toomanycodons]
nbrMutCodonsTot <- nbrMutCodonsTot[!toomanycodons]
nbrMutCodons <- nbrMutCodons[!toomanycodons]
nbrMutBases <- S4Vectors::elementNROWS(unique(mismatchPositions[!toomanycodons]))
## Forbidden codons
uniqueMutCodons <- unique(codonMismatches)
mutCodons <- S4Vectors::endoapply(uniqueMutCodons,
function(v) rep(3 * v, each = 3) + c(-2, -1, 0))
mutCodons <- as(varReverse, "DNAStringSet")[mutCodons]
matchForbidden <- Biostrings::vmatchPattern(
pattern = Ldef$forbiddenMutatedCodonsReverse,
as(mutCodons, "DNAStringSet"),
fixed = FALSE)
forbiddencodon <- sapply(BiocGenerics::relist(start(unlist(matchForbidden)) %% 3==1,
matchForbidden), function(i) any(i))
message("Number of reads with forbidden codons, reverse: ", sum(forbiddencodon)) ## 2
fq1 <- fq1[!forbiddencodon]
fq2 <- fq2[!forbiddencodon]
encodedMutCodonsForward <- encodedMutCodonsForward[!forbiddencodon]
mutCodons <- mutCodons[!forbiddencodon]
nbrMutCodonsTot <- nbrMutCodonsTot[!forbiddencodon]
nbrMutBasesTot <- nbrMutBasesTot[!forbiddencodon]
nbrMutCodons <- nbrMutCodons[!forbiddencodon]
nbrMutBases <- nbrMutBases[!forbiddencodon]
uniqueMutCodons <- uniqueMutCodons[!forbiddencodon]
splitMutCodons <- as(strsplit(gsub("([^.]{3})", "\\1\\.",
as.character(mutCodons)), ".", fixed = TRUE),
"CharacterList")
encodedMutCodonsReverse <- S4Vectors::unstrsplit(BiocGenerics::relist(
paste0("r", unlist(uniqueMutCodons), unlist(splitMutCodons)),
uniqueMutCodons),
sep = "_")
mutNames <- paste(encodedMutCodonsForward, encodedMutCodonsReverse, sep = "_")
nbrMutCodonsTot <- nbrMutCodonsTot + nbrMutCodons
nbrMutBasesTot <- nbrMutBasesTot + nbrMutBases
} else {
mutNames <- encodedMutCodonsForward
}
## Constant sequence filtering
keep <- rep(TRUE, length(fq1))
if (!all(Ldef$constantForward == "") && Ldef$constantMaxDistForward > (-1)) {
constForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward + 1,
width = Ldef$constantLengthForward)
dists <- as.matrix(pwalign::stringDist(
c(DNAStringSet(structure(Ldef$constantForward,
names = paste0("C", length(Ldef$constantForward)))),
DNAStringSet(constForward)
)
))
dists <- dists[-(1:length(Ldef$constantForward)), 1:length(Ldef$constantForward), drop = FALSE]
keep <- keep & (apply(dists, 1, min) <= Ldef$constantMaxDistForward)
}
if (!all(Ldef$constantReverse == "") && Ldef$constantMaxDistReverse > (-1)) {
constReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse + 1,
width = Ldef$constantLengthReverse)
dists <- as.matrix(pwalign::stringDist(
c(DNAStringSet(structure(Ldef$constantReverse,
names = paste0("C", length(Ldef$constantReverse)))),
DNAStringSet(constReverse)
)
))
dists <- dists[-(1:length(Ldef$constantReverse)), 1:length(Ldef$constantReverse), drop = FALSE]
keep <- keep & (apply(dists, 1, min) <= Ldef$constantMaxDistReverse)
}
message("Number of reads with too many mismatches in constant seq: ", sum(!keep))
fq1 <- fq1[keep]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[keep]
}
mutCodons <- mutCodons[keep]
uniqueMutCodons <- uniqueMutCodons[keep]
mutNames <- mutNames[keep]
if (!is.null(nbrMutBasesTot)) {
nbrMutBasesTot <- nbrMutBasesTot[keep]
nbrMutCodonsTot <- nbrMutCodonsTot[keep]
## Number of mutated bases and codons
varForwardFinal <- Biostrings::subseq(
fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
if (!is.null(Ldef$fastqReverse)) {
varReverseFinal <- Biostrings::subseq(
fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
} else {
varReverseFinal <- ""
}
message("Number of mutated codons per retained read: ")
print(table(nbrMutCodonsTot))
message("Number of different reads with each number of mismatched codons: ")
print(table(nbrMutCodonsTot[!duplicated(paste0(varForwardFinal, "_", varReverseFinal))]))
message("Number of mutated bases per retained read: ")
print(table(nbrMutBasesTot))
message("Number of different reads with each number of mismatched bases: ")
print(table(nbrMutBasesTot[!duplicated(paste0(varForwardFinal, "_", varReverseFinal))]))
## Number of mutated amino acids
## Forward
p_aa <- Biostrings::translate(DNAStringSet(rep(Ldef$wildTypeForward, length(varForwardFinal))))
pattern_width_aa <- width(p_aa)
varForwardFinal_aa <- Biostrings::translate(varForwardFinal)
subject_width_aa <- width(varForwardFinal_aa)
unlisted_ans_aa <- which(as.raw(unlist(p_aa)) != as.raw(unlist(varForwardFinal_aa)))
breakpoints_aa <- cumsum(pattern_width_aa)
ans_eltlens_aa <- tabulate(findInterval(unlisted_ans_aa - 1L,
breakpoints_aa) + 1L,
nbins = length(p_aa))
skeleton_aa <- IRanges::PartitioningByEnd(cumsum(ans_eltlens_aa))
aaMismatchesForward <- BiocGenerics::relist(unlisted_ans_aa, skeleton_aa)
## Reverse
if (!is.null(Ldef$fastqReverse)) {
p_aa <- Biostrings::translate(DNAStringSet(rep(Ldef$wildTypeReverse, length(varReverseFinal))))
pattern_width_aa <- width(p_aa)
varReverseFinal_aa <- Biostrings::translate(varReverseFinal)
subject_width_aa <- width(varReverseFinal_aa)
unlisted_ans_aa <- which(as.raw(unlist(p_aa)) != as.raw(unlist(varReverseFinal_aa)))
breakpoints_aa <- cumsum(pattern_width_aa)
ans_eltlens_aa <- tabulate(findInterval(unlisted_ans_aa - 1L,
breakpoints_aa) + 1L,
nbins = length(p_aa))
skeleton_aa <- IRanges::PartitioningByEnd(cumsum(ans_eltlens_aa))
aaMismatchesReverse <- BiocGenerics::relist(unlisted_ans_aa, skeleton_aa)
message("Number of mutated aas per retained read: ")
print(table(lengths(aaMismatchesForward) + lengths(aaMismatchesReverse)))
message("Number of different reads with each number of mismatched aas: ")
print(table((lengths(aaMismatchesForward) + lengths(aaMismatchesReverse))[!duplicated(paste0(varForwardFinal, "_", varReverseFinal))]))
} else {
message("Number of mutated aas per retained read: ")
print(table(lengths(aaMismatchesForward)))
message("Number of different reads with each number of mismatched aas: ")
print(table(lengths(aaMismatchesForward)[!duplicated(varForwardFinal)]))
}
}
mutNames <- gsub("^_", "", gsub("_$", "", mutNames))
mutNames[mutNames == ""] <- "WT"
message("Number of variants seen twice: ", sum(table(mutNames) == 2)) ## 2
message("Variants seen twice: ")
print(which(table(mutNames) == 2))
## Retained reads
message("Number of retained reads: ", length(fq1)) ## 279
## Error statistics
## Forward
if (length(Ldef$constantForward) == 1) {
constForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward + 1,
width = Ldef$constantLengthForward)
p <- DNAStringSet(rep(Ldef$constantForward, length(constForward)))
pattern_width <- width(p)
subject_width <- width(constForward)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(constForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
mismatchQualities <- unlist(as(quality(constForward)[mismatchPositions], "IntegerList"))
unlisted_ans <- which(as.raw(unlist(p)) == as.raw(unlist(constForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
matchPositions <- nucleotideMatches - offsets
matchQualities <- unlist(as(quality(constForward)[matchPositions], "IntegerList"))
message("Match qualities, forward:")
print(table(matchQualities))
message("Mismatch qualities, forward:")
print(table(mismatchQualities))
}
## Reverse
if (!is.null(Ldef$fastqReverse) && length(Ldef$constantReverse) == 1) {
constReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse + 1,
width = Ldef$constantLengthReverse)
#constReverse <- Biostrings::reverseComplement(constReverse)
p <- DNAStringSet(rep(Ldef$constantReverse, length(constReverse)))
pattern_width <- width(p)
subject_width <- width(constReverse)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(constReverse)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
mismatchQualities <- unlist(as(quality(constReverse)[mismatchPositions], "IntegerList"))
unlisted_ans <- which(as.raw(unlist(p)) == as.raw(unlist(constReverse)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
matchPositions <- nucleotideMatches - offsets
matchQualities <- unlist(as(quality(constReverse)[matchPositions], "IntegerList"))
message("Match qualities, reverse:")
print(table(matchQualities))
message("Mismatch qualities, reverse:")
print(table(mismatchQualities))
}
## Collapsing
if (Ldef$wildTypeForward == "" && Ldef$wildTypeReverse == "") {
umicoll <- list()
if (!is.null(Ldef$fastqReverse)) {
fqseq <- paste0(as.character(varForward), "_", as.character(varReverse))
} else {
fqseq <- as.character(varForward)
}
tbl <- as.data.frame(table(fqseq)) %>% dplyr::arrange(desc(Freq), fqseq) %>%
dplyr::mutate(fqseq = as.character(fqseq))
tbl$umis <- ""
for (i in seq_len(nrow(tbl))) {
tbl$umis[i] <- paste(umis[fqseq == tbl$fqseq[i]], collapse = ",")
}
collseqs <- collapse_seqs(tbl$fqseq, tbl$umis, tbl$Freq,
maxdist = Ldef$variableCollapseMaxDist,
umimaxdist = Ldef$umiCollapseMaxDist,
minreads = Ldef$variableCollapseMinReads,
minratio = Ldef$variableCollapseMinRatio)
message("Number of unique sequences: ", nrow(tbl))
message("Number of collapsed sequences: ", length(collseqs))
message("Total UMI count: ", length(unlist(collseqs)))
return(tbl)
}
return(NULL)
}
## ----------------------------------------------------------------------------
## Generate values for comparison in unit tests - trans experiment
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
## ----------------------------------------------------------------------------
## Change some parameter values
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 30.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNA",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 25.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
## ----------------------------------------------------------------------------
## Only one input file
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
Ldef <- list(
fastqForward = fqt1, fastqReverse = NULL,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 30.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNA",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 25.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
## Test collapsing
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
maxNReads = -1, variableCollapseMaxDist = 6, umiCollapseMaxDist = 4,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = NULL,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
variableCollapseMaxDist = 10, umiCollapseMaxDist = 5,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
maxNReads = -1, verbose = FALSE
)
processReadsTrans(Ldef)
## don't add anything between the above and below runs
## same as above but with new collapsing approach
Ldef$variableCollapseMaxDist <- 0
Ldef$umiCollapseMaxDist <- 5
Ldef$variableCollapseMinReads <- 0
Ldef$variableCollapseMinRatio <- 0
v <- processReadsTrans(Ldef)
cseq <- collapse_seqs(v$fqseq, v$umis, v$Freq, maxdist = 10,
umimaxdist = 0, minreads = 0, minratio = 0)
length(cseq)
## Collapse only highly abundant features
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
maxNReads = -1, variableCollapseMaxDist = 2, umiCollapseMaxDist = 0,
variableCollapseMinReads = 2, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
v <- processReadsTrans(Ldef)
## don't add anything between the above and below runs
## same as above but with new collapsing approach
Ldef$variableCollapseMaxDist <- 0
Ldef$umiCollapseMaxDist <- 0
Ldef$variableCollapseMinReads <- 0
Ldef$variableCollapseMinRatio <- 0
v <- processReadsTrans(Ldef)
cseq <- collapse_seqs(v$fqseq, v$umis, v$Freq, maxdist = 2,
umimaxdist = 0, minreads = 1.5, minratio = 0)
length(cseq)
## Collapse only features with high enough count ratio
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
maxNReads = -1, variableCollapseMaxDist = 3, umiCollapseMaxDist = 0,
variableCollapseMinReads = 1, variableCollapseMinRatio = 1.5,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
v <- processReadsTrans(Ldef)
## don't add anything between the above and below runs
## same as above but with new collapsing approach
Ldef$variableCollapseMaxDist <- 0
Ldef$umiCollapseMaxDist <- 0
Ldef$variableCollapseMinReads <- 0
Ldef$variableCollapseMinRatio <- 0
v <- processReadsTrans(Ldef)
cseq <- collapse_seqs(v$fqseq, v$umis, v$Freq, maxdist = 3,
umimaxdist = 0, minreads = 1, minratio = 1.5)
length(cseq)
## filter based on constant sequences
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = 0, constantMaxDistReverse = 0,
verbose = FALSE
)
processReadsTrans(Ldef)
## 1 mismatch
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = 1, constantMaxDistReverse = 1,
verbose = FALSE
)
processReadsTrans(Ldef)
## Multiple constant sequences
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = c("AACCGGAGGAGGGAGCTG", "AACCGGCGGAGGGAGCTG"),
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = 0, constantMaxDistReverse = 0,
verbose = FALSE
)
processReadsTrans(Ldef)
fmicompbio/mutscan documentation built on Oct. 24, 2024, 2:41 p.m.
R Package Documentation
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suppressPackageStartupMessages({
library(Biostrings)
library(ShortRead)
library(dplyr)
})
collapse_seqs <- function(seqs, umis, reads, maxdist, umimaxdist, minreads, minratio) {
tokeep <- list()
while (length(seqs) > 0) {
if (reads[1] >= minreads) {
d <- stringdist::stringdist(seqs[1], seqs, method = "hamming")
w <- union(1, which(d <= maxdist & reads[1] >= minratio * reads))
} else {
w <- 1
}
tokeep[[seqs[1]]] <- paste(umis[w], collapse = ",")
seqs <- seqs[-w]
umis <- umis[-w]
reads <- reads[-w]
}
tokeep <- lapply(tokeep, function(tk) {
tk <- sort(unique(strsplit(tk, ",")[[1]]))
if (length(tk) == 1) {
tk
} else {
k <- c()
while (length(tk) > 0) {
d <- stringdist::stringdist(tk[1], tk, method = "hamming")
w <- which(d <= umimaxdist)
k <- c(k, tk[1])
tk <- tk[-w]
}
k
}
})
tokeep
}
processReadsTrans <- function(Ldef) {
## Read data
fq1 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqForward)
if (!is.null(Ldef$fastqReverse)) {
fq2 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqReverse)
}
## Number of reads
message("Number of reads: ", length(fq1)) ## 1000
## Adapter sequences
adapt <- Biostrings::vcountPattern(Ldef$adapterForward, fq1) > 0
if (!is.null(Ldef$fastqReverse)) {
adapt2 <- Biostrings::vcountPattern(Ldef$adapterReverse, fq2) > 0
adapt <- adapt | adapt2
}
message("Number of reads with adapters: ", sum(adapt)) ## 314
fq1 <- fq1[!adapt]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!adapt]
}
## Average quality in variable sequences too low
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
avgQualForward <- ShortRead::alphabetScore(Biostrings::quality(varForward))/
BiocGenerics::width(varForward)
lowqual <- avgQualForward < Ldef$avePhredMinForward
if (!is.null(Ldef$fastqReverse)) {
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
avgQualReverse <- ShortRead::alphabetScore(Biostrings::quality(varReverse))/
BiocGenerics::width(varReverse)
lowqual <- lowqual | avgQualReverse < Ldef$avePhredMinReverse
}
message("Number of reads with low average quality: ", sum(lowqual)) # 7
fq1 <- fq1[!lowqual]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!lowqual]
}
## Too many Ns in variable sequences
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
nbrNForward <- Biostrings::vcountPattern("N", varForward, fixed = TRUE)
toomanynvar <- nbrNForward > Ldef$variableNMaxForward
if (!is.null(Ldef$fastqReverse)) {
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
nbrNReverse <- Biostrings::vcountPattern("N", varReverse, fixed = TRUE)
toomanynvar <- toomanynvar | nbrNReverse > Ldef$variableNMaxReverse
}
message("Number of reads with too many N in variable sequence: ", sum(toomanynvar)) # 0
fq1 <- fq1[!toomanynvar]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanynvar]
}
## Too many Ns in UMIs
umiForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + 1, width = Ldef$umiLengthForward)
if (!is.null(Ldef$fastqReverse)) {
umiReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + 1, width = Ldef$umiLengthReverse)
umis <- Biostrings::xscat(umiForward, umiReverse)
} else {
umis <- umiForward
}
nbrNumi <- Biostrings::vcountPattern("N", umis, fixed = TRUE)
toomanynumi <- nbrNumi > Ldef$umiNMax
message("Number of reads with too many N in UMI: ", sum(toomanynumi)) # 0
fq1 <- fq1[!toomanynumi]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanynumi]
}
## Compare to wildtype, forward
mutCodons <- NULL
uniqueMutCodons <- NULL
mutBases <- NULL
uniqueMutBases <- NULL
nbrMutBasesTot <- NULL
if (Ldef$wildTypeForward != "") {
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
p <- DNAStringSet(rep(Ldef$wildTypeForward, length(varForward)))
pattern_width <- width(p)
subject_width <- width(varForward)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(varForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
## First check, excluding reads with more than 3x(max nbr mut codons) mismatches
baseMismatches <- mismatchPositions
nbrMutBases <- S4Vectors::elementNROWS(unique(baseMismatches))
toomanybases <- nbrMutBases > 3 * Ldef$nbrMutatedCodonsMaxForward
message("Number of reads with too many mutated codons, forward (1): ", sum(toomanybases))
fq1 <- fq1[!toomanybases]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanybases]
}
mismatchPositions <- mismatchPositions[!toomanybases]
varForward <- varForward[!toomanybases]
mismatchQualities <- Biostrings::quality(varForward)[mismatchPositions]
lowqualmm <- min(as(mismatchQualities, "IntegerList")) < Ldef$mutatedPhredMinForward
message("Number of reads with low-quality mismatch bases, forward: ", sum(lowqualmm)) ## 0
fq1 <- fq1[!lowqualmm]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!lowqualmm]
}
mismatchPositions <- mismatchPositions[!lowqualmm]
varForward <- varForward[!lowqualmm]
## Number of mutated codons
codonMismatches <- (mismatchPositions - 1) %/% 3 + 1
nbrMutCodons <- S4Vectors::elementNROWS(unique(codonMismatches))
toomanycodons <- nbrMutCodons > Ldef$nbrMutatedCodonsMaxForward
message("Number of reads with too many mutated codons, forward: ", sum(toomanycodons)) ## 287
fq1 <- fq1[!toomanycodons]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanycodons]
}
codonMismatches <- codonMismatches[!toomanycodons]
varForward <- varForward[!toomanycodons]
nbrMutCodons <- nbrMutCodons[!toomanycodons]
nbrMutBases <- S4Vectors::elementNROWS(unique(mismatchPositions[!toomanycodons]))
## Forbidden codons
uniqueMutCodons <- unique(codonMismatches)
mutCodons <- S4Vectors::endoapply(uniqueMutCodons,
function(v) rep(3 * v, each = 3) + c(-2, -1, 0))
mutCodons <- as(varForward, "DNAStringSet")[mutCodons]
matchForbidden <- Biostrings::vmatchPattern(
pattern = Ldef$forbiddenMutatedCodonsForward,
as(mutCodons, "DNAStringSet"),
fixed = FALSE)
forbiddencodon <- sapply(BiocGenerics::relist(start(unlist(matchForbidden)) %% 3==1,
matchForbidden), function(i) any(i))
message("Number of reads with forbidden codons, forward: ", sum(forbiddencodon)) ## 6
fq1 <- fq1[!forbiddencodon]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!forbiddencodon]
}
mutCodons <- mutCodons[!forbiddencodon]
nbrMutCodonsTot <- nbrMutCodons[!forbiddencodon]
nbrMutBasesTot <- nbrMutBases[!forbiddencodon]
uniqueMutCodons <- uniqueMutCodons[!forbiddencodon]
splitMutCodons <- as(strsplit(gsub("([^.]{3})", "\\1\\.",
as.character(mutCodons)), ".", fixed = TRUE),
"CharacterList")
encodedMutCodonsForward <- S4Vectors::unstrsplit(BiocGenerics::relist(
paste0("f", unlist(uniqueMutCodons), unlist(splitMutCodons)),
uniqueMutCodons),
sep = "_")
} else {
encodedMutCodonsForward <- ""
}
## Compare to wildtype, reverse
if (!is.null(Ldef$fastqReverse) && Ldef$wildTypeReverse != "") {
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
p <- DNAStringSet(rep(Ldef$wildTypeReverse, length(varReverse)))
pattern_width <- width(p)
subject_width <- width(varReverse)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(varReverse)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
## First check, excluding reads with more than 3x(max nbr mut codons) mismatches
baseMismatches <- mismatchPositions
nbrMutBases <- S4Vectors::elementNROWS(unique(baseMismatches))
toomanybases <- nbrMutBases > 3 * Ldef$nbrMutatedCodonsMaxReverse
message("Number of reads with too many mutated codons, reverse (1): ", sum(toomanybases))
fq1 <- fq1[!toomanybases]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[!toomanybases]
}
mismatchPositions <- mismatchPositions[!toomanybases]
varReverse <- varReverse[!toomanybases]
encodedMutCodonsForward <- encodedMutCodonsForward[!toomanybases]
nbrMutBasesTot <- nbrMutBasesTot[!toomanybases]
nbrMutCodonsTot <- nbrMutCodonsTot[!toomanybases]
mismatchQualities <- Biostrings::quality(varReverse)[mismatchPositions]
lowqualmm <- min(as(mismatchQualities, "IntegerList")) < Ldef$mutatedPhredMinReverse
message("Number of reads with low-quality mismatch bases, reverse: ", sum(lowqualmm)) ## 0
fq1 <- fq1[!lowqualmm]
fq2 <- fq2[!lowqualmm]
mismatchPositions <- mismatchPositions[!lowqualmm]
varReverse <- varReverse[!lowqualmm]
encodedMutCodonsForward <- encodedMutCodonsForward[!lowqualmm]
nbrMutBasesTot <- nbrMutBasesTot[!lowqualmm]
nbrMutCodonsTot <- nbrMutCodonsTot[!lowqualmm]
## Number of mutated codons
codonMismatches <- (mismatchPositions - 1) %/% 3 + 1
nbrMutCodons <- S4Vectors::elementNROWS(unique(codonMismatches))
toomanycodons <- nbrMutCodons > Ldef$nbrMutatedCodonsMaxReverse
message("Number of reads with too many mutated codons, reverse: ", sum(toomanycodons)) ## 105
fq1 <- fq1[!toomanycodons]
fq2 <- fq2[!toomanycodons]
codonMismatches <- codonMismatches[!toomanycodons]
varReverse <- varReverse[!toomanycodons]
encodedMutCodonsForward <- encodedMutCodonsForward[!toomanycodons]
nbrMutBasesTot <- nbrMutBasesTot[!toomanycodons]
nbrMutCodonsTot <- nbrMutCodonsTot[!toomanycodons]
nbrMutCodons <- nbrMutCodons[!toomanycodons]
nbrMutBases <- S4Vectors::elementNROWS(unique(mismatchPositions[!toomanycodons]))
## Forbidden codons
uniqueMutCodons <- unique(codonMismatches)
mutCodons <- S4Vectors::endoapply(uniqueMutCodons,
function(v) rep(3 * v, each = 3) + c(-2, -1, 0))
mutCodons <- as(varReverse, "DNAStringSet")[mutCodons]
matchForbidden <- Biostrings::vmatchPattern(
pattern = Ldef$forbiddenMutatedCodonsReverse,
as(mutCodons, "DNAStringSet"),
fixed = FALSE)
forbiddencodon <- sapply(BiocGenerics::relist(start(unlist(matchForbidden)) %% 3==1,
matchForbidden), function(i) any(i))
message("Number of reads with forbidden codons, reverse: ", sum(forbiddencodon)) ## 2
fq1 <- fq1[!forbiddencodon]
fq2 <- fq2[!forbiddencodon]
encodedMutCodonsForward <- encodedMutCodonsForward[!forbiddencodon]
mutCodons <- mutCodons[!forbiddencodon]
nbrMutCodonsTot <- nbrMutCodonsTot[!forbiddencodon]
nbrMutBasesTot <- nbrMutBasesTot[!forbiddencodon]
nbrMutCodons <- nbrMutCodons[!forbiddencodon]
nbrMutBases <- nbrMutBases[!forbiddencodon]
uniqueMutCodons <- uniqueMutCodons[!forbiddencodon]
splitMutCodons <- as(strsplit(gsub("([^.]{3})", "\\1\\.",
as.character(mutCodons)), ".", fixed = TRUE),
"CharacterList")
encodedMutCodonsReverse <- S4Vectors::unstrsplit(BiocGenerics::relist(
paste0("r", unlist(uniqueMutCodons), unlist(splitMutCodons)),
uniqueMutCodons),
sep = "_")
mutNames <- paste(encodedMutCodonsForward, encodedMutCodonsReverse, sep = "_")
nbrMutCodonsTot <- nbrMutCodonsTot + nbrMutCodons
nbrMutBasesTot <- nbrMutBasesTot + nbrMutBases
} else {
mutNames <- encodedMutCodonsForward
}
## Constant sequence filtering
keep <- rep(TRUE, length(fq1))
if (!all(Ldef$constantForward == "") && Ldef$constantMaxDistForward > (-1)) {
constForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward + 1,
width = Ldef$constantLengthForward)
dists <- as.matrix(pwalign::stringDist(
c(DNAStringSet(structure(Ldef$constantForward,
names = paste0("C", length(Ldef$constantForward)))),
DNAStringSet(constForward)
)
))
dists <- dists[-(1:length(Ldef$constantForward)), 1:length(Ldef$constantForward), drop = FALSE]
keep <- keep & (apply(dists, 1, min) <= Ldef$constantMaxDistForward)
}
if (!all(Ldef$constantReverse == "") && Ldef$constantMaxDistReverse > (-1)) {
constReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse + 1,
width = Ldef$constantLengthReverse)
dists <- as.matrix(pwalign::stringDist(
c(DNAStringSet(structure(Ldef$constantReverse,
names = paste0("C", length(Ldef$constantReverse)))),
DNAStringSet(constReverse)
)
))
dists <- dists[-(1:length(Ldef$constantReverse)), 1:length(Ldef$constantReverse), drop = FALSE]
keep <- keep & (apply(dists, 1, min) <= Ldef$constantMaxDistReverse)
}
message("Number of reads with too many mismatches in constant seq: ", sum(!keep))
fq1 <- fq1[keep]
if (!is.null(Ldef$fastqReverse)) {
fq2 <- fq2[keep]
}
mutCodons <- mutCodons[keep]
uniqueMutCodons <- uniqueMutCodons[keep]
mutNames <- mutNames[keep]
if (!is.null(nbrMutBasesTot)) {
nbrMutBasesTot <- nbrMutBasesTot[keep]
nbrMutCodonsTot <- nbrMutCodonsTot[keep]
## Number of mutated bases and codons
varForwardFinal <- Biostrings::subseq(
fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
if (!is.null(Ldef$fastqReverse)) {
varReverseFinal <- Biostrings::subseq(
fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
} else {
varReverseFinal <- ""
}
message("Number of mutated codons per retained read: ")
print(table(nbrMutCodonsTot))
message("Number of different reads with each number of mismatched codons: ")
print(table(nbrMutCodonsTot[!duplicated(paste0(varForwardFinal, "_", varReverseFinal))]))
message("Number of mutated bases per retained read: ")
print(table(nbrMutBasesTot))
message("Number of different reads with each number of mismatched bases: ")
print(table(nbrMutBasesTot[!duplicated(paste0(varForwardFinal, "_", varReverseFinal))]))
## Number of mutated amino acids
## Forward
p_aa <- Biostrings::translate(DNAStringSet(rep(Ldef$wildTypeForward, length(varForwardFinal))))
pattern_width_aa <- width(p_aa)
varForwardFinal_aa <- Biostrings::translate(varForwardFinal)
subject_width_aa <- width(varForwardFinal_aa)
unlisted_ans_aa <- which(as.raw(unlist(p_aa)) != as.raw(unlist(varForwardFinal_aa)))
breakpoints_aa <- cumsum(pattern_width_aa)
ans_eltlens_aa <- tabulate(findInterval(unlisted_ans_aa - 1L,
breakpoints_aa) + 1L,
nbins = length(p_aa))
skeleton_aa <- IRanges::PartitioningByEnd(cumsum(ans_eltlens_aa))
aaMismatchesForward <- BiocGenerics::relist(unlisted_ans_aa, skeleton_aa)
## Reverse
if (!is.null(Ldef$fastqReverse)) {
p_aa <- Biostrings::translate(DNAStringSet(rep(Ldef$wildTypeReverse, length(varReverseFinal))))
pattern_width_aa <- width(p_aa)
varReverseFinal_aa <- Biostrings::translate(varReverseFinal)
subject_width_aa <- width(varReverseFinal_aa)
unlisted_ans_aa <- which(as.raw(unlist(p_aa)) != as.raw(unlist(varReverseFinal_aa)))
breakpoints_aa <- cumsum(pattern_width_aa)
ans_eltlens_aa <- tabulate(findInterval(unlisted_ans_aa - 1L,
breakpoints_aa) + 1L,
nbins = length(p_aa))
skeleton_aa <- IRanges::PartitioningByEnd(cumsum(ans_eltlens_aa))
aaMismatchesReverse <- BiocGenerics::relist(unlisted_ans_aa, skeleton_aa)
message("Number of mutated aas per retained read: ")
print(table(lengths(aaMismatchesForward) + lengths(aaMismatchesReverse)))
message("Number of different reads with each number of mismatched aas: ")
print(table((lengths(aaMismatchesForward) + lengths(aaMismatchesReverse))[!duplicated(paste0(varForwardFinal, "_", varReverseFinal))]))
} else {
message("Number of mutated aas per retained read: ")
print(table(lengths(aaMismatchesForward)))
message("Number of different reads with each number of mismatched aas: ")
print(table(lengths(aaMismatchesForward)[!duplicated(varForwardFinal)]))
}
}
mutNames <- gsub("^_", "", gsub("_$", "", mutNames))
mutNames[mutNames == ""] <- "WT"
message("Number of variants seen twice: ", sum(table(mutNames) == 2)) ## 2
message("Variants seen twice: ")
print(which(table(mutNames) == 2))
## Retained reads
message("Number of retained reads: ", length(fq1)) ## 279
## Error statistics
## Forward
if (length(Ldef$constantForward) == 1) {
constForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward + 1,
width = Ldef$constantLengthForward)
p <- DNAStringSet(rep(Ldef$constantForward, length(constForward)))
pattern_width <- width(p)
subject_width <- width(constForward)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(constForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
mismatchQualities <- unlist(as(quality(constForward)[mismatchPositions], "IntegerList"))
unlisted_ans <- which(as.raw(unlist(p)) == as.raw(unlist(constForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
matchPositions <- nucleotideMatches - offsets
matchQualities <- unlist(as(quality(constForward)[matchPositions], "IntegerList"))
message("Match qualities, forward:")
print(table(matchQualities))
message("Mismatch qualities, forward:")
print(table(mismatchQualities))
}
## Reverse
if (!is.null(Ldef$fastqReverse) && length(Ldef$constantReverse) == 1) {
constReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse + 1,
width = Ldef$constantLengthReverse)
#constReverse <- Biostrings::reverseComplement(constReverse)
p <- DNAStringSet(rep(Ldef$constantReverse, length(constReverse)))
pattern_width <- width(p)
subject_width <- width(constReverse)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(constReverse)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
mismatchQualities <- unlist(as(quality(constReverse)[mismatchPositions], "IntegerList"))
unlisted_ans <- which(as.raw(unlist(p)) == as.raw(unlist(constReverse)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
matchPositions <- nucleotideMatches - offsets
matchQualities <- unlist(as(quality(constReverse)[matchPositions], "IntegerList"))
message("Match qualities, reverse:")
print(table(matchQualities))
message("Mismatch qualities, reverse:")
print(table(mismatchQualities))
}
## Collapsing
if (Ldef$wildTypeForward == "" && Ldef$wildTypeReverse == "") {
umicoll <- list()
if (!is.null(Ldef$fastqReverse)) {
fqseq <- paste0(as.character(varForward), "_", as.character(varReverse))
} else {
fqseq <- as.character(varForward)
}
tbl <- as.data.frame(table(fqseq)) %>% dplyr::arrange(desc(Freq), fqseq) %>%
dplyr::mutate(fqseq = as.character(fqseq))
tbl$umis <- ""
for (i in seq_len(nrow(tbl))) {
tbl$umis[i] <- paste(umis[fqseq == tbl$fqseq[i]], collapse = ",")
}
collseqs <- collapse_seqs(tbl$fqseq, tbl$umis, tbl$Freq,
maxdist = Ldef$variableCollapseMaxDist,
umimaxdist = Ldef$umiCollapseMaxDist,
minreads = Ldef$variableCollapseMinReads,
minratio = Ldef$variableCollapseMinRatio)
message("Number of unique sequences: ", nrow(tbl))
message("Number of collapsed sequences: ", length(collseqs))
message("Total UMI count: ", length(unlist(collseqs)))
return(tbl)
}
return(NULL)
}
## ----------------------------------------------------------------------------
## Generate values for comparison in unit tests - trans experiment
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
## ----------------------------------------------------------------------------
## Change some parameter values
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 30.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNA",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 25.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
## ----------------------------------------------------------------------------
## Only one input file
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
Ldef <- list(
fastqForward = fqt1, fastqReverse = NULL,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 30.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNA",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 25.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
## Test collapsing
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
maxNReads = -1, variableCollapseMaxDist = 6, umiCollapseMaxDist = 4,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
processReadsTrans(Ldef)
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = NULL,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
variableCollapseMaxDist = 10, umiCollapseMaxDist = 5,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
maxNReads = -1, verbose = FALSE
)
processReadsTrans(Ldef)
## don't add anything between the above and below runs
## same as above but with new collapsing approach
Ldef$variableCollapseMaxDist <- 0
Ldef$umiCollapseMaxDist <- 5
Ldef$variableCollapseMinReads <- 0
Ldef$variableCollapseMinRatio <- 0
v <- processReadsTrans(Ldef)
cseq <- collapse_seqs(v$fqseq, v$umis, v$Freq, maxdist = 10,
umimaxdist = 0, minreads = 0, minratio = 0)
length(cseq)
## Collapse only highly abundant features
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
maxNReads = -1, variableCollapseMaxDist = 2, umiCollapseMaxDist = 0,
variableCollapseMinReads = 2, variableCollapseMinRatio = 0,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
v <- processReadsTrans(Ldef)
## don't add anything between the above and below runs
## same as above but with new collapsing approach
Ldef$variableCollapseMaxDist <- 0
Ldef$umiCollapseMaxDist <- 0
Ldef$variableCollapseMinReads <- 0
Ldef$variableCollapseMinRatio <- 0
v <- processReadsTrans(Ldef)
cseq <- collapse_seqs(v$fqseq, v$umis, v$Freq, maxdist = 2,
umimaxdist = 0, minreads = 1.5, minratio = 0)
length(cseq)
## Collapse only features with high enough count ratio
fqt1 <- system.file("extdata/transInput_1.fastq.gz", package = "mutscan")
fqt2 <- system.file("extdata/transInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE,
minOverlap = 0, maxOverlap = 0, maxFracMismatchOverlap = 0, greedyOverlap = TRUE,
revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
primerForward = "",
primerReverse = "",
wildTypeForward = "",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
mutNameDelimiter = ".",
maxNReads = -1, variableCollapseMaxDist = 3, umiCollapseMaxDist = 0,
variableCollapseMinReads = 1, variableCollapseMinRatio = 1.5,
constantMaxDistForward = -1, constantMaxDistReverse = -1,
verbose = FALSE
)
v <- processReadsTrans(Ldef)
## don't add anything between the above and below runs
## same as above but with new collapsing approach
Ldef$variableCollapseMaxDist <- 0
Ldef$umiCollapseMaxDist <- 0
Ldef$variableCollapseMinReads <- 0
Ldef$variableCollapseMinRatio <- 0
v <- processReadsTrans(Ldef)
cseq <- collapse_seqs(v$fqseq, v$umis, v$Freq, maxdist = 3,
umimaxdist = 0, minreads = 1, minratio = 1.5)
length(cseq)
## filter based on constant sequences
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = 0, constantMaxDistReverse = 0,
verbose = FALSE
)
processReadsTrans(Ldef)
## 1 mismatch
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = 1, constantMaxDistReverse = 1,
verbose = FALSE
)
processReadsTrans(Ldef)
## Multiple constant sequences
Ldef <- list(
fastqForward = fqt1, fastqReverse = fqt2,
mergeForwardReverse = FALSE, revComplForward = FALSE, revComplReverse = FALSE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 8,
constantLengthForward = 18, constantLengthReverse = 20,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "ATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTT",
constantForward = c("AACCGGAGGAGGGAGCTG", "AACCGGCGGAGGGAGCTG"),
constantReverse = "GAAAAAGGAAGCTGGAGAGA",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = 1,
nbrMutatedCodonsMaxReverse = 1,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
variableCollapseMaxDist = 0, umiCollapseMaxDist = 0,
variableCollapseMinReads = 0, variableCollapseMinRatio = 0,
constantMaxDistForward = 0, constantMaxDistReverse = 0,
verbose = FALSE
)
processReadsTrans(Ldef)
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