inst/scripts/generateValuesForUnitTestsCis.R
In fmicompbio/mutscan: Preprocessing and Analysis of Deep Mutational Scanning Data
suppressPackageStartupMessages({
library(Biostrings)
library(ShortRead)
})
processReadsCis <- function(Ldef) {
## Read data
fq1 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqForward)
fq2 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqReverse)
## Number of reads
message("Number of reads: ", length(fq1)) ## 1000
## Adapter sequences
adapt <- Biostrings::vcountPattern(Ldef$adapterForward, fq1) > 0 |
Biostrings::vcountPattern(Ldef$adapterReverse, fq2) > 0
message("Number of reads with adapters: ", sum(adapt)) ## 126
fq1 <- fq1[!adapt]
fq2 <- fq2[!adapt]
## Merge sequences
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
varReverse <- Biostrings::reverseComplement(varReverse)
replaceAt <- as(ShortRead::FastqQuality(Biostrings::quality(varReverse)), "matrix") >
as(ShortRead::FastqQuality(Biostrings::quality(varForward)), "matrix")
replaceAtList <- as(lapply(seq_len(nrow(replaceAt)),
function(x) which(replaceAt[x, ])), "IntegerList")
consensusSeq <- Biostrings::replaceLetterAt(x = varForward, at = replaceAt,
letter = varReverse[replaceAtList])
qualsForward <- as(ShortRead::FastqQuality(Biostrings::quality(varForward)), "matrix")
qualsReverse <- as(ShortRead::FastqQuality(Biostrings::quality(varReverse)), "matrix")
qualsForward[replaceAt] <- qualsReverse[replaceAt]
phredQuals <- apply(qualsForward, 1, function(v) seqTools::ascii2char(v + 33))
consensusQuals <- Biostrings::PhredQuality(phredQuals)
varForward <- Biostrings::QualityScaledDNAStringSet(consensusSeq, consensusQuals)
## Average quality in variable sequences too low
avgQualForward <- ShortRead::alphabetScore(Biostrings::quality(varForward))/
BiocGenerics::width(varForward)
lowqual <- avgQualForward < Ldef$avePhredMinForward
message("Number of reads with low average quality: ", sum(lowqual)) # 0
fq1 <- fq1[!lowqual]
fq2 <- fq2[!lowqual]
varForward <- varForward[!lowqual]
## Too many Ns in variable sequences
nbrNForward <- Biostrings::vcountPattern("N", varForward, fixed = TRUE)
toomanynvar <- nbrNForward > Ldef$variableNMaxForward
message("Number of reads with too many N in variable sequence: ", sum(toomanynvar)) # 44
fq1 <- fq1[!toomanynvar]
fq2 <- fq2[!toomanynvar]
varForward <- varForward[!toomanynvar]
## Too many Ns in UMIs
umiForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + 1, width = Ldef$umiLengthForward)
umiReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + 1, width = Ldef$umiLengthReverse)
umis <- Biostrings::xscat(umiForward, umiReverse)
nbrNumi <- Biostrings::vcountPattern("N", umis, fixed = TRUE)
toomanynumi <- nbrNumi > Ldef$umiNMax
message("Number of reads with too many N in UMI: ", sum(toomanynumi)) # 0
fq1 <- fq1[!toomanynumi]
fq2 <- fq2[!toomanynumi]
varForward <- varForward[!toomanynumi]
## Compare to wildtype, forward
p <- DNAStringSet(rep(Ldef$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
mismatchQualities <- Biostrings::quality(varForward)[mismatchPositions]
lowqualmm <- min(as(mismatchQualities, "IntegerList")) < Ldef$mutatedPhredMinForward
message("Number of reads with low-quality mismatch bases: ", sum(lowqualmm)) ## 0
fq1 <- fq1[!lowqualmm]
fq2 <- fq2[!lowqualmm]
mismatchPositions <- mismatchPositions[!lowqualmm]
varForward <- varForward[!lowqualmm]
## Number of mutated codons/bases
codonMismatches <- (mismatchPositions - 1) %/% 3 + 1
nbrMutCodons <- S4Vectors::elementNROWS(unique(codonMismatches))
baseMismatches <- mismatchPositions
nbrMutBases <- S4Vectors::elementNROWS(unique(baseMismatches))
if (Ldef$nbrMutatedCodonsMaxForward != (-1)) {
toomanycodons <- nbrMutCodons > Ldef$nbrMutatedCodonsMaxForward
} else {
toomanycodons <- nbrMutBases > Ldef$nbrMutatedBasesMaxForward
}
message("Number of reads with too many mutated codons: ", sum(toomanycodons)) ## 581
fq1 <- fq1[!toomanycodons]
fq2 <- fq2[!toomanycodons]
codonMismatches <- codonMismatches[!toomanycodons]
nbrMutCodons <- nbrMutCodons[!toomanycodons]
baseMismatches <- baseMismatches[!toomanycodons]
nbrMutBases <- nbrMutBases[!toomanycodons]
varForward <- varForward[!toomanycodons]
## Forbidden codons
# if (Ldef$nbrMutatedCodonsMaxForward != (-1)) {
uniqueMutCodons <- unique(codonMismatches)
uniqueMutBases <- unique(baseMismatches)
mutBases <- S4Vectors::endoapply(uniqueMutBases,
function(v) v)
mutBases <- as(varForward, "DNAStringSet")[mutBases]
mutCodons <- S4Vectors::endoapply(uniqueMutCodons,
function(v) rep(3 * v, each = 3) + c(-2, -1, 0))
mutCodons <- as(varForward, "DNAStringSet")[mutCodons]
matchForbidden <- Biostrings::vmatchPattern(
pattern = Ldef$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: ", sum(forbiddencodon)) ## 82
fq1 <- fq1[!forbiddencodon]
fq2 <- fq2[!forbiddencodon]
varForward <- varForward[!forbiddencodon]
mutCodons <- mutCodons[!forbiddencodon]
mutBases <- mutBases[!forbiddencodon]
uniqueMutCodons <- uniqueMutCodons[!forbiddencodon]
uniqueMutBases <- uniqueMutBases[!forbiddencodon]
nbrMutCodons <- nbrMutCodons[!forbiddencodon]
nbrMutBases <- nbrMutBases[!forbiddencodon]
if (Ldef$nbrMutatedCodonsMaxForward != (-1)) {
splitMutCodons <- as(strsplit(gsub("([^.]{3})", "\\1\\.",
as.character(mutCodons)), ".", fixed = TRUE),
"CharacterList")
encodedMutCodonsForward <- S4Vectors::unstrsplit(BiocGenerics::relist(
paste0("f", unlist(uniqueMutCodons), unlist(splitMutCodons)),
uniqueMutCodons),
sep = "_")
} else {
splitMutBases <- as(strsplit(gsub("([^.]{1})", "\\1\\.",
as.character(mutBases)), ".", fixed = TRUE),
"CharacterList")
encodedMutCodonsForward <- S4Vectors::unstrsplit(BiocGenerics::relist(
paste0("f", unlist(uniqueMutBases), unlist(splitMutBases)),
uniqueMutBases),
sep = "_")
}
mutNames <- encodedMutCodonsForward
mutNames <- gsub("^_", "", gsub("_$", "", mutNames))
mutNames[mutNames <- ""] <- "WT"
message("Number of variants seen thrice: ", sum(table(mutNames) == 3)) ## 5
message("Variants seen thrice: ")
print(which(table(mutNames) == 3))
## Retained reads
message("Number of retained reads: ", length(fq1)) ## 167
## Number of mutated bases and codons
message("Number of mutated codons per retained read: ")
print(table(nbrMutCodons))
message("Number of different reads with each number of mismatched codons: ")
print(table(nbrMutCodons[!duplicated(varForward)]))
message("Number of mutated bases per retained read: ")
print(table(nbrMutBases))
message("Number of different reads with each number of mismatched bases: ")
print(table(nbrMutBases[!duplicated(varForward)]))
## Number of mutated amino acids
p_aa <- Biostrings::translate(DNAStringSet(rep(Ldef$wildTypeForward, length(varForward))))
pattern_width_aa <- width(p_aa)
varForward_aa <- Biostrings::translate(varForward)
subject_width_aa <- width(varForward_aa)
unlisted_ans_aa <- which(as.raw(unlist(p_aa)) != as.raw(unlist(varForward_aa)))
breakpoints_aa <- cumsum(pattern_width_aa)
ans_eltlens_aa <- tabulate(findInterval(unlisted_ans_aa - 1L,
breakpoints_aa) + 1L,
nbins = length(p_aa))
skeleton_aa <- IRanges::PartitioningByEnd(cumsum(ans_eltlens_aa))
aaMismatches <- BiocGenerics::relist(unlisted_ans_aa, skeleton_aa)
message("Number of mutated aas per retained read: ")
print(table(lengths(aaMismatches))) ## 97, 70
message("Number of different reads with each number of mismatched aas: ")
print(table(lengths(aaMismatches)[!duplicated(varForward)])) ## 14, 53
## Error statistics
## Forward
constForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward + 1,
width = Ldef$constantLengthForward)
p <- DNAStringSet(rep(Ldef$constantForward, length(constForward)))
pattern_width <- width(p)
subject_width <- width(constForward)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(constForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
mismatchQualities <- unlist(as(quality(constForward)[mismatchPositions], "IntegerList"))
unlisted_ans <- which(as.raw(unlist(p)) == as.raw(unlist(constForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
matchPositions <- nucleotideMatches - offsets
matchQualities <- unlist(as(quality(constForward)[matchPositions], "IntegerList"))
message("Match qualities, forward:")
print(table(matchQualities))
message("Mismatch qualities, forward:")
print(table(mismatchQualities))
## Reverse
constReverse <- Biostrings::subseq(fq2, start = Ldef$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))
}
## ----------------------------------------------------------------------------
## Generate values for comparison in unit tests - cis experiment
fqc1 <- system.file("extdata/cisInput_1.fastq.gz", package = "mutscan")
fqc2 <- system.file("extdata/cisInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqc1, fastqReverse = fqc2,
mergeForwardReverse = TRUE, revComplForward = FALSE, revComplReverse = TRUE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 7,
constantLengthForward = 18, constantLengthReverse = 17,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAGTTCATCCTGGCAGC",
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,
verbose = FALSE
)
processReadsCis(Ldef)
## Limit number of mismatching bases rather than codons
Ldef <- list(
fastqForward = fqc1, fastqReverse = fqc2,
mergeForwardReverse = TRUE, revComplForward = FALSE, revComplReverse = TRUE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 7,
constantLengthForward = 18, constantLengthReverse = 17,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAGTTCATCCTGGCAGC",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = -1,
nbrMutatedCodonsMaxReverse = -1,
nbrMutatedBasesMaxForward = 2,
nbrMutatedBasesMaxReverse = 2,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
verbose = FALSE
)
processReadsCis(Ldef)
fmicompbio/mutscan documentation built on Oct. 24, 2024, 2:41 p.m.
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suppressPackageStartupMessages({
library(Biostrings)
library(ShortRead)
})
processReadsCis <- function(Ldef) {
## Read data
fq1 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqForward)
fq2 <- Biostrings::readQualityScaledDNAStringSet(Ldef$fastqReverse)
## Number of reads
message("Number of reads: ", length(fq1)) ## 1000
## Adapter sequences
adapt <- Biostrings::vcountPattern(Ldef$adapterForward, fq1) > 0 |
Biostrings::vcountPattern(Ldef$adapterReverse, fq2) > 0
message("Number of reads with adapters: ", sum(adapt)) ## 126
fq1 <- fq1[!adapt]
fq2 <- fq2[!adapt]
## Merge sequences
varForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + Ldef$umiLengthForward +
Ldef$constantLengthForward + 1, width = Ldef$variableLengthForward)
varReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + Ldef$umiLengthReverse +
Ldef$constantLengthReverse + 1, width = Ldef$variableLengthReverse)
varReverse <- Biostrings::reverseComplement(varReverse)
replaceAt <- as(ShortRead::FastqQuality(Biostrings::quality(varReverse)), "matrix") >
as(ShortRead::FastqQuality(Biostrings::quality(varForward)), "matrix")
replaceAtList <- as(lapply(seq_len(nrow(replaceAt)),
function(x) which(replaceAt[x, ])), "IntegerList")
consensusSeq <- Biostrings::replaceLetterAt(x = varForward, at = replaceAt,
letter = varReverse[replaceAtList])
qualsForward <- as(ShortRead::FastqQuality(Biostrings::quality(varForward)), "matrix")
qualsReverse <- as(ShortRead::FastqQuality(Biostrings::quality(varReverse)), "matrix")
qualsForward[replaceAt] <- qualsReverse[replaceAt]
phredQuals <- apply(qualsForward, 1, function(v) seqTools::ascii2char(v + 33))
consensusQuals <- Biostrings::PhredQuality(phredQuals)
varForward <- Biostrings::QualityScaledDNAStringSet(consensusSeq, consensusQuals)
## Average quality in variable sequences too low
avgQualForward <- ShortRead::alphabetScore(Biostrings::quality(varForward))/
BiocGenerics::width(varForward)
lowqual <- avgQualForward < Ldef$avePhredMinForward
message("Number of reads with low average quality: ", sum(lowqual)) # 0
fq1 <- fq1[!lowqual]
fq2 <- fq2[!lowqual]
varForward <- varForward[!lowqual]
## Too many Ns in variable sequences
nbrNForward <- Biostrings::vcountPattern("N", varForward, fixed = TRUE)
toomanynvar <- nbrNForward > Ldef$variableNMaxForward
message("Number of reads with too many N in variable sequence: ", sum(toomanynvar)) # 44
fq1 <- fq1[!toomanynvar]
fq2 <- fq2[!toomanynvar]
varForward <- varForward[!toomanynvar]
## Too many Ns in UMIs
umiForward <- Biostrings::subseq(fq1, start = Ldef$skipForward + 1, width = Ldef$umiLengthForward)
umiReverse <- Biostrings::subseq(fq2, start = Ldef$skipReverse + 1, width = Ldef$umiLengthReverse)
umis <- Biostrings::xscat(umiForward, umiReverse)
nbrNumi <- Biostrings::vcountPattern("N", umis, fixed = TRUE)
toomanynumi <- nbrNumi > Ldef$umiNMax
message("Number of reads with too many N in UMI: ", sum(toomanynumi)) # 0
fq1 <- fq1[!toomanynumi]
fq2 <- fq2[!toomanynumi]
varForward <- varForward[!toomanynumi]
## Compare to wildtype, forward
p <- DNAStringSet(rep(Ldef$wildTypeForward, length(varForward)))
pattern_width <- width(p)
subject_width <- width(varForward)
unlisted_ans <- which(as.raw(unlist(p)) != as.raw(unlist(varForward)))
breakpoints <- cumsum(pattern_width)
ans_eltlens <- tabulate(findInterval(unlisted_ans - 1L,
breakpoints) + 1L,
nbins = length(p))
skeleton <- IRanges::PartitioningByEnd(cumsum(ans_eltlens))
nucleotideMismatches <- BiocGenerics::relist(unlisted_ans, skeleton)
offsets <- c(0L, breakpoints[-length(breakpoints)])
mismatchPositions <- nucleotideMismatches - offsets
mismatchQualities <- Biostrings::quality(varForward)[mismatchPositions]
lowqualmm <- min(as(mismatchQualities, "IntegerList")) < Ldef$mutatedPhredMinForward
message("Number of reads with low-quality mismatch bases: ", sum(lowqualmm)) ## 0
fq1 <- fq1[!lowqualmm]
fq2 <- fq2[!lowqualmm]
mismatchPositions <- mismatchPositions[!lowqualmm]
varForward <- varForward[!lowqualmm]
## Number of mutated codons/bases
codonMismatches <- (mismatchPositions - 1) %/% 3 + 1
nbrMutCodons <- S4Vectors::elementNROWS(unique(codonMismatches))
baseMismatches <- mismatchPositions
nbrMutBases <- S4Vectors::elementNROWS(unique(baseMismatches))
if (Ldef$nbrMutatedCodonsMaxForward != (-1)) {
toomanycodons <- nbrMutCodons > Ldef$nbrMutatedCodonsMaxForward
} else {
toomanycodons <- nbrMutBases > Ldef$nbrMutatedBasesMaxForward
}
message("Number of reads with too many mutated codons: ", sum(toomanycodons)) ## 581
fq1 <- fq1[!toomanycodons]
fq2 <- fq2[!toomanycodons]
codonMismatches <- codonMismatches[!toomanycodons]
nbrMutCodons <- nbrMutCodons[!toomanycodons]
baseMismatches <- baseMismatches[!toomanycodons]
nbrMutBases <- nbrMutBases[!toomanycodons]
varForward <- varForward[!toomanycodons]
## Forbidden codons
# if (Ldef$nbrMutatedCodonsMaxForward != (-1)) {
uniqueMutCodons <- unique(codonMismatches)
uniqueMutBases <- unique(baseMismatches)
mutBases <- S4Vectors::endoapply(uniqueMutBases,
function(v) v)
mutBases <- as(varForward, "DNAStringSet")[mutBases]
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$forbiddenMutatedCodonsForward,
as(mutCodons, "DNAStringSet"),
fixed = FALSE)
forbiddencodon <- sapply(BiocGenerics::relist(start(unlist(matchForbidden)) %% 3==1,
matchForbidden), function(i) any(i))
message("Number of reads with forbidden codons: ", sum(forbiddencodon)) ## 82
fq1 <- fq1[!forbiddencodon]
fq2 <- fq2[!forbiddencodon]
varForward <- varForward[!forbiddencodon]
mutCodons <- mutCodons[!forbiddencodon]
mutBases <- mutBases[!forbiddencodon]
uniqueMutCodons <- uniqueMutCodons[!forbiddencodon]
uniqueMutBases <- uniqueMutBases[!forbiddencodon]
nbrMutCodons <- nbrMutCodons[!forbiddencodon]
nbrMutBases <- nbrMutBases[!forbiddencodon]
if (Ldef$nbrMutatedCodonsMaxForward != (-1)) {
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 {
splitMutBases <- as(strsplit(gsub("([^.]{1})", "\\1\\.",
as.character(mutBases)), ".", fixed = TRUE),
"CharacterList")
encodedMutCodonsForward <- S4Vectors::unstrsplit(BiocGenerics::relist(
paste0("f", unlist(uniqueMutBases), unlist(splitMutBases)),
uniqueMutBases),
sep = "_")
}
mutNames <- encodedMutCodonsForward
mutNames <- gsub("^_", "", gsub("_$", "", mutNames))
mutNames[mutNames <- ""] <- "WT"
message("Number of variants seen thrice: ", sum(table(mutNames) == 3)) ## 5
message("Variants seen thrice: ")
print(which(table(mutNames) == 3))
## Retained reads
message("Number of retained reads: ", length(fq1)) ## 167
## Number of mutated bases and codons
message("Number of mutated codons per retained read: ")
print(table(nbrMutCodons))
message("Number of different reads with each number of mismatched codons: ")
print(table(nbrMutCodons[!duplicated(varForward)]))
message("Number of mutated bases per retained read: ")
print(table(nbrMutBases))
message("Number of different reads with each number of mismatched bases: ")
print(table(nbrMutBases[!duplicated(varForward)]))
## Number of mutated amino acids
p_aa <- Biostrings::translate(DNAStringSet(rep(Ldef$wildTypeForward, length(varForward))))
pattern_width_aa <- width(p_aa)
varForward_aa <- Biostrings::translate(varForward)
subject_width_aa <- width(varForward_aa)
unlisted_ans_aa <- which(as.raw(unlist(p_aa)) != as.raw(unlist(varForward_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))
aaMismatches <- BiocGenerics::relist(unlisted_ans_aa, skeleton_aa)
message("Number of mutated aas per retained read: ")
print(table(lengths(aaMismatches))) ## 97, 70
message("Number of different reads with each number of mismatched aas: ")
print(table(lengths(aaMismatches)[!duplicated(varForward)])) ## 14, 53
## Error statistics
## Forward
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
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))
}
## ----------------------------------------------------------------------------
## Generate values for comparison in unit tests - cis experiment
fqc1 <- system.file("extdata/cisInput_1.fastq.gz", package = "mutscan")
fqc2 <- system.file("extdata/cisInput_2.fastq.gz", package = "mutscan")
## default arguments
Ldef <- list(
fastqForward = fqc1, fastqReverse = fqc2,
mergeForwardReverse = TRUE, revComplForward = FALSE, revComplReverse = TRUE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 7,
constantLengthForward = 18, constantLengthReverse = 17,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAGTTCATCCTGGCAGC",
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,
verbose = FALSE
)
processReadsCis(Ldef)
## Limit number of mismatching bases rather than codons
Ldef <- list(
fastqForward = fqc1, fastqReverse = fqc2,
mergeForwardReverse = TRUE, revComplForward = FALSE, revComplReverse = TRUE,
skipForward = 1, skipReverse = 1,
umiLengthForward = 10, umiLengthReverse = 7,
constantLengthForward = 18, constantLengthReverse = 17,
variableLengthForward = 96, variableLengthReverse = 96,
adapterForward = "GGAAGAGCACACGTC",
adapterReverse = "GGAAGAGCGTCGTGT",
wildTypeForward = "ACTGATACACTCCAAGCGGAGACAGACCAACTAGAAGATGAGAAGTCTGCTTTGCAGACCGAGATTGCCAACCTGCTGAAGGAGAAGGAAAAACTA",
wildTypeReverse = "",
constantForward = "AACCGGAGGAGGGAGCTG",
constantReverse = "GAGTTCATCCTGGCAGC",
avePhredMinForward = 20.0, avePhredMinReverse = 20.0,
variableNMaxForward = 0, variableNMaxReverse = 0,
umiNMax = 0,
nbrMutatedCodonsMaxForward = -1,
nbrMutatedCodonsMaxReverse = -1,
nbrMutatedBasesMaxForward = 2,
nbrMutatedBasesMaxReverse = 2,
forbiddenMutatedCodonsForward = "NNW",
forbiddenMutatedCodonsReverse = "NNW",
mutatedPhredMinForward = 0.0, mutatedPhredMinReverse = 0.0,
verbose = FALSE
)
processReadsCis(Ldef)
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