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inst/tests/test-hic-pair.R
In diffHic: Differential Analyis of Hi-C Data
###################################################################################################
# This script is designed to test the pair-identifying capabilities of the hiC machinery i.e. preparePairs.
# We start with unit tests for individual components of the preparePairs C++ code.
suppressPackageStartupMessages(require(diffHic))
suppressPackageStartupMessages(require(GenomicAlignments))
source("simsam.R")
dir<-"hic-test"
dir.create(dir)
# Checking CIGAR.
checkCIGAR <- function(cigar, rstrand) {
output <- simsam(file.path(dir, "whee"), "chrA", 1, !rstrand, c("chrA"=1000),
cigar=cigar, len=cigarWidthAlongQuerySpace(cigar))
out <- .Call(diffHic:::cxx_test_parse_cigar, output)
if (is.character(out)) { stop(out) }
true.alen <- cigarWidthAlongReferenceSpace(cigar)
if (out[1]!=true.alen) { stop("mismatch in alignment length") }
offset <- 0
as.rle <- cigarToRleList(cigar)[[1]]
if (rstrand) { as.rle <- rev(as.rle) }
if (runValue(as.rle)[1]=="H") { offset <- runLength(as.rle)[1] }
if (out[2]!=offset) { stop("mismatch in offsets") }
return(c(alen=true.alen, offset=offset))
}
checkCIGAR("5H20M", TRUE)
checkCIGAR("5H20M", FALSE)
checkCIGAR("5H20M6H", TRUE)
checkCIGAR("5H20M6H", FALSE)
checkCIGAR("5H20M6S", TRUE)
checkCIGAR("5H20M6S", FALSE)
checkCIGAR("20M5I30M", TRUE)
checkCIGAR("20M5D30M", TRUE)
checkCIGAR("20M5N30M", TRUE)
checkCIGAR("20M5P30M", TRUE)
checkCIGAR("20M5X30M", TRUE)
checkCIGAR("20M5=30M", TRUE)
checkCIGAR("10M2I5M3D20M", TRUE)
checkCIGAR("10M2I3D5N20M", TRUE)
checkCIGAR("10M2P5M3D20I", TRUE)
checkCIGAR("10M2I3D5N20D", TRUE)
checkCIGAR("5H11M5D8I9X6=6H", TRUE)
checkCIGAR("5H20X5D8P9N72M", FALSE)
checkCIGAR("5S19M34=55D8X20M6S", TRUE)
checkCIGAR("5S1M3=5D18X2M6S", FALSE)
# Checking fragment assignment.
assign2fragment <- function(starts, ends, chr, pos, rstrand, len) {
out <- .Call(diffHic:::cxx_test_fragment_assign, starts, ends, chr, pos, rstrand, len)
if (is.character(out)) { stop(out) }
chr <- chr + 1L
if (rstrand) {
fiveprime <- min(pos + len -1L, tail(ends[[chr]], 1))
stopifnot(ends[[chr]][out] >= fiveprime && (out==1L || ends[[chr]][out-1] < fiveprime))
} else {
fiveprime <- pos
stopifnot(starts[[chr]][out] <= fiveprime && (out==length(starts[[chr]]) || starts[[chr]][out+1] > fiveprime))
}
out
}
starts <- list( c(1L, 100L, 200L, 300L, 400L, 500L), # chr1
c(1L, 100L, 200L, 300L, 400L, 500L)) # chr2
ends <- list( c(103L, 203L, 303L, 403L, 503L, 1000L), # chr1
c(103L, 203L, 303L, 403L, 503L, 1000L)) # chr2
assign2fragment(starts, ends, 0L, 94L, TRUE, 10L)
assign2fragment(starts, ends, 0L, 95L, TRUE, 10L)
assign2fragment(starts, ends, 0L, 99L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 100L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 203L, TRUE, 1L)
assign2fragment(starts, ends, 0L, 204L, TRUE, 1L)
assign2fragment(starts, ends, 0L, 209L, FALSE, 1L)
assign2fragment(starts, ends, 0L, 300L, FALSE, 1L)
assign2fragment(starts, ends, 1L, 1L, FALSE, 10L)
assign2fragment(starts, ends, 1L, 991L, FALSE, 10L)
assign2fragment(starts, ends, 1L, 992L, FALSE, 10L)
assign2fragment(starts, ends, 1L, 991L, TRUE, 10L)
assign2fragment(starts, ends, 1L, 992L, TRUE, 10L)
starts <- list( c(1L, 1L, 100L, 200L, 300L, 400L, 500L, 997L), # chr1, with nesting at the start and end.
c(1L, 1L, 100L, 200L, 300L, 400L, 500L, 997L)) # chr2
ends <- list( c(4L, 103L, 203L, 303L, 403L, 503L, 1000L, 1000L), # chr1
c(4L, 103L, 203L, 303L, 403L, 503L, 1000L, 1000L)) # chr2
assign2fragment(starts, ends, 0L, 1L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 1L, TRUE, 10L)
assign2fragment(starts, ends, 0L, 991L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 991L, TRUE, 10L)
try(assign2fragment(starts, ends, 0L, 1000L, TRUE, 10L)) # This should fail, as it gets assigned into the nested fragment.
###################################################################################################
# We also set up a full simulation for the entire function.
suppressPackageStartupMessages(require("rhdf5"))
comp <- function (fname, npairs, max.cuts, sizes=c(100, 500), singles=0, rlen=10, overhang=0L, pseudo=FALSE, extras=NULL, storage=5000) {
################ SETTING UP THE FRAGMENTS ###################
rlen <- as.integer(rlen)
overhang <- as.integer(overhang)
if (min(sizes) <= rlen) { stop("min fragment must be greater than read length") }
# Necessary for proper assignment, especially at the start of the chromosome when reverse
# reads are bounded at zero (i.e. their 5' ends would not be defined if 1+rlen > fragmentsize)
# Randomly generating fragment lengths for the chromosome.
fragments <- cut.starts <- outfrags <- list()
chromosomes <- integer(length(max.cuts))
for (i in seq_along(max.cuts)) {
fragments[[i]] <- as.integer(round(runif(max.cuts[[i]], sizes[1], sizes[2])))
ends <- cumsum(fragments[[i]]) - seq_len(max.cuts[[i]])*overhang
if (max.cuts[[i]] > 1L) {
cut.starts[[i]] <- c(1L, (ends+1L)[seq_len(max.cuts[[i]]-1)])
} else {
cut.starts[[i]] <- 1L
}
outfrags[[i]] <- GRanges(names(max.cuts)[i], IRanges(cut.starts[[i]], ends+overhang))
chromosomes[i] <- tail(ends, 1)+overhang
}
seq.names <- names(max.cuts)
seq.lengths <- chromosomes
suppressWarnings(outfrags <- do.call(c, outfrags))
seqlevels(outfrags) <- seq.names
seqlengths(outfrags) <- setNames(seq.lengths, seq.names)
names(chromosomes) <- names(max.cuts)
names(fragments) <- names(max.cuts)
names(cut.starts) <- names(max.cuts)
################ SETTING UP THE READ PAIRS ###################
# Randomly generating reads (a la getPESizes' example).
names <- paste('x', rep(seq_len(npairs), 2), sep=".")
chrs <- sample(length(chromosomes), length(names), replace=TRUE)
pos <- integer(length(names));
frag.ids <- integer(length(names))
str <- rbinom(length(names), 1, 0.5)==1
# Assigning positions to all of them. Some finesse is necessary when
# there is no overhang (i.e. original genome with positive overhang). We
# still allow reads to span restriction sites, though.
for (i in seq_along(chromosomes)) {
current <- chrs==i
chosen.frags <- as.integer(runif(sum(current), 1, length(fragments[[i]])+1))
frag.ids[current] <- chosen.frags
my.ends <- cut.starts[[i]] + fragments[[i]]
# Note that as.integer(runif(1, a, b)) samples from [a, b), as runif() will never actually generate 'b'.
# So, this will only generate positions after and including the start position for the chosen fragment,
# but before and not including the start position for the next fragment (or 1-past the end of the chromosome).
cur.for <- str[current]
forward.frag <- chosen.frags[cur.for]
forward.min <- cut.starts[[i]][forward.frag]
forward.max <- ifelse(forward.frag!=length(cut.starts[[i]]), cut.starts[[i]][forward.frag+1L], my.ends[forward.frag])
pos[current][cur.for] <- as.integer(runif(sum(cur.for), forward.min, forward.max))
# Recall that the 'ends' are 1-past the last base of the fragment, so this runif() will sample from
# the first non-overlapping base of the current fragment to the last base of the current fragment
# (it's [a, b), so when b is 1-past the last base, sampling will include the last base).
reverse.frag <- chosen.frags[!cur.for]
reverse.min <- integer(sum(!cur.for))
possible.zero <- reverse.frag==1L
reverse.min[possible.zero] <- 1L
reverse.min[!possible.zero] <- my.ends[reverse.frag[!possible.zero]-1L]
reverse.max <- my.ends[reverse.frag]
pos[current][!cur.for] <- pmax(1L, as.integer(runif(sum(!cur.for), reverse.min, reverse.max)) - rlen + 1L)
}
# Throwing them into the SAM file generator. Note that chromosome names are ordered inside.
# If this differs from the order in 'max.cuts', it will test the ability of preparePairs to match them up correctly.
reversi <- c(seq_len(npairs) + npairs, seq_len(npairs))
out <- simsam(fname, names(chromosomes)[chrs], pos, str, chromosomes, names=names, len=rlen,
is.first=rep(c(TRUE, FALSE), each=npairs), is.paired=TRUE,
mate.chr=names(chromosomes)[chrs][reversi], mate.pos=pos[reversi], mate.str=str[reversi])
if (singles) {
# Adding some singles. You'll get some warnings regarding overhang regions as we're not finessing it.
snames <- schrs <- spos <- NULL
snames <- paste('y', seq_len(singles), sep=".")
schrs <- sample(length(chromosomes), singles, replace=TRUE)
spos <- integer(singles);
for (i in seq_along(chromosomes)) {
scurrent <- schrs==i;
spos[scurrent] <- as.integer(round(runif(sum(scurrent), 1, chromosomes[i])))
}
tempname <- file.path(dir, "temp")
sstr <- rbinom(singles, 1, 0.5)==1
out2 <- simsam(tempname, names(chromosomes)[schrs], spos, sstr, chromosomes, names=snames, len=rlen)
more.temp <- file.path(dir, "temp2")
out <- mergeBam(c(out, out2), more.temp, indexDestination=TRUE, overwrite=TRUE)
file.rename(more.temp, out)
}
# Resorting by name.
temp <- sortBam(out, "temp", byQname=TRUE)
file.rename(temp, out)
################ THEORETICAL MATCH ###################
# This gets the status: 0 for okay, 1 for dangling end, 3 for self.circles and and 2 for other stuff.
getstatus <- function(chr1, pos1, frag1, str1, chr2, pos2, frag2, str2) {
codes <- integer(length(chr1))
potentials <- chr1==chr2 & frag1==frag2
same.str <- str1==str2;
codes[ same.str & potentials ] <- 2L
potentials <- potentials & !same.str
self.circle <- (str1 & pos2+rlen <= pos1) | (str2 & pos1+rlen <= pos2);
codes[ self.circle & potentials ] <- 3L
potentials <- potentials & !self.circle
# Not worrying about overextension; these are now considered to be dangling ends,
# as sequencing/trimming/alignment errors can often result in a few overextended bases.
# overextension <- (str1 & pos1 > pos2) | (str2 & pos2 > pos1) # Don't need to check +rlen, as they're al the same.
# codes[ overextension & potentials ] <- 2L
# potentials <- potentials & !overextension
codes[potentials] <- 1L
return(codes)
}
# This gets the distance from the 5' end of the read to the next restriction site that it is pointing to (past the one it covers, if it is incomplete).
getlen <- function(chr, pos, start, str) {
len.out <- integer(length(chr))
for (x in seq_along(cut.starts)) {
chosen <- x==chr
dist2cut <- ifelse(str[chosen], cut.starts[[x]][start[chosen]]+fragments[[x]][start[chosen]]-pos[chosen],
pos[chosen]+rlen-cut.starts[[x]][start[chosen]])
len.out[chosen] <- dist2cut
}
return(len.out)
}
# Now, actually assembling the theoretical values.
primary <- seq_len(npairs)
secondary<- npairs + primary
pchrs <- chrs[primary]
ppos <- pos[primary]
pfrag <- frag.ids[primary]
pstr <- str[primary]
schrs <- chrs[secondary]
spos <- pos[secondary]
sfrag <- frag.ids[secondary]
sstr <- str[secondary]
codes <- getstatus(pchrs, ppos, pfrag, pstr, schrs, spos, sfrag, sstr)
if (pseudo) {
frag.lens <- rep(NA_integer_, npairs)
pfrag[] <- 0L
sfrag[] <- 0L
} else {
frag.lens <- getlen(pchrs, ppos, pfrag, pstr) + getlen(schrs, spos, sfrag, sstr)
}
ppos5 <- ifelse(pstr, ppos, ppos+rlen -1L)
spos5 <- ifelse(sstr, spos, spos+rlen -1L)
panchor <- pchrs > schrs | (pchrs==schrs & pfrag > sfrag) | (pchrs==schrs & pfrag==sfrag & ppos5 > spos5)
inserts <- ifelse(pchrs==schrs, pmax(ppos, spos)-pmin(spos, ppos)+rlen, NA)
orientations <- ifelse(pstr, 0L, ifelse(panchor, 1L, 2L))+ifelse(sstr, 0L, ifelse(panchor, 2L, 1L))
################ ACTUAL MATCH ###################
# Assembling the output list for comparison.
# Adding an extra chromosome to the start, to see if the match is still correct when it's not 1:1.
# Also deleting a chromosome to trigger an error.
if (!is.null(extras)) {
if (extras[1]==-1L) {
used.frags <- dropSeqlevels(outfrags, seqnames(outfrags)[1], pruning.mode="coarse")
} else {
used.frags <- suppressWarnings(c(GRanges(extras, IRanges(1, 100)), outfrags))
}
} else {
used.frags <- outfrags
}
tmpdir<-paste0(fname, "_temp")
param <- pairParam(fragments=used.frags)
if (pseudo) {
# Special behaviour; faster assignment into bins, no removal of dangling ends/self-cirlces
# (as these concepts are meaningless for arbitrary bins).
param <- pairParam(used.frags[0])
diagnostics <- preparePairs(out, param, tmpdir, output.dir=file.path(dir, "whee"), storage=storage)
} else {
diagnostics <- preparePairs(out, param, tmpdir, output.dir=file.path(dir, "whee"), storage=storage)
stopifnot(sum(codes==1L)==diagnostics$same.id[["dangling"]])
stopifnot(sum(codes==3L)==diagnostics$same.id[["self.circle"]])
stopifnot(length(codes)==diagnostics$pairs[["total"]])
stopifnot(singles==diagnostics$singles)
# No support for testing chimeras, we use a fixed example below.
stopifnot(diagnostics$unmapped.chimeras==0L)
stopifnot(diagnostics$chimeras[["total"]]==0L)
stopifnot(diagnostics$chimeras[["mapped"]]==0L)
stopifnot(diagnostics$chimeras[["invalid"]]==0L)
}
# Anchor1/anchor2 synchronisation is determined by order in 'fragments' (and thusly, in max.cuts).
if (pseudo) {
offset <- integer(length(chromosomes)) # 'extras' don't matter, as ID is set to zero anyway.
} else {
offset <- c(0L, cumsum(max.cuts))
names(offset) <- NULL
offset <- offset + length(extras)
}
indices <- diffHic:::preloader(tmpdir)
fchrs <- as.character(seqnames(used.frags))
for (i in seq_along(max.cuts)) {
for (j in seq_len(i)) {
# Getting the fragment IDs for the remaining pairs for this chromosome.
stuff <- (pchrs==i & schrs==j) | (pchrs==j & schrs==i)
if (!pseudo) { stuff <- stuff & (codes==0L | codes==2L) }
pids <- pfrag[stuff]
sids <- sfrag[stuff]
pps <- ppos[stuff]
sps <- spos[stuff]
pls <- ifelse(pstr[stuff], 1L, -1L)*rlen
sls <- ifelse(sstr[stuff], 1L, -1L)*rlen
which.is.which <- panchor[stuff]
anchor1 <- ifelse(which.is.which, pids, sids) + offset[i]
anchor2 <- ifelse(which.is.which, sids, pids) + offset[j]
ap1 <- ifelse(which.is.which, pps, sps)
ap2 <- ifelse(which.is.which, sps, pps)
al1 <- ifelse(which.is.which, pls, sls)
al2 <- ifelse(which.is.which, sls, pls)
totes <- frag.lens[stuff]
cur.ori <- orientations[stuff]
cur.insert <- inserts[stuff]
o <- order(anchor1, anchor2, ap1, ap2, al1, al2, totes, cur.ori, cur.insert)
# Extracting data from HDF5 and computing statistics.
achr <- names(max.cuts)[i]
tchr <- names(max.cuts)[j]
if (!(achr%in%names(indices)) || !(tchr %in% names(indices[[achr]]))) {
if (length(o)) { stop("true interactions are missing") }
next
}
current <- h5read(tmpdir, file.path(achr, tchr))
for (x in seq_len(ncol(current))) { attributes(current[,x]) <- NULL }
collated <- diffHic:::.getStats(current, achr==tchr, used.frags)
# Checking anchor1/anchor2/length/orientation/insert statistics (sorting on everything to ensure comparability).
o2 <- order(current$anchor1.id, current$anchor2.id, current$anchor1.pos, current$anchor2.pos,
current$anchor1.len, current$anchor2.len, collated$length, collated$orientation, collated$insert)
stopifnot(identical(current$anchor1.id[o2], anchor1[o]))
stopifnot(identical(current$anchor2.id[o2], anchor2[o]))
stopifnot(identical(current$anchor1.pos[o2], ap1[o]))
stopifnot(identical(current$anchor2.pos[o2], ap2[o]))
stopifnot(identical(current$anchor1.len[o2], al1[o]))
stopifnot(identical(current$anchor2.len[o2], al2[o]))
stopifnot(identical(collated$length[o2], totes[o]))
stopifnot(identical(collated$orientation[o2], cur.ori[o]))
stopifnot(identical(collated$insert[o2], cur.insert[o]))
# Checking that choice and ordering of anchor1 and anchor2 is sane.
current5.1 <- current$anchor1.pos - pmin(0L, current$anchor1.len + 1L)
current5.2 <- current$anchor2.pos - pmin(0L, current$anchor2.len + 1L)
if (!pseudo) {
stopifnot(all(current$anchor1.id >= current$anchor2.id))
stopifnot(all((current5.1 >= current5.2)[current$anchor1.id==current$anchor2.id]))
o <- order(current$anchor1.id, current$anchor2.id)
stopifnot(!is.unsorted(o))
} else {
stopifnot(all(current$anchor1.id==0L & current$anchor2.id==0L))
if (i==j) {
if (!all(current5.1 >= current5.2)) {
print(current)
}
stopifnot(all(current5.1 >= current5.2))
}
}
# Checking that we're looking at the right combination.
if (!pseudo) {
uniq.a <- unique(fchrs[current$anchor1.id])
uniq.t <- unique(fchrs[current$anchor2.id])
if (length(uniq.a)!=1L || length(uniq.t)!=1L) { stop("file contains more than one combination") }
if (achr!=uniq.a || tchr!=uniq.t) { stop("file contains the incorrect combination") }
}
# Wiping it out to indicate that we've visited this place.
indices[[achr]][[tchr]]<-NULL
}
}
# Checking there's nothing left.
if (!is.null(unlist(indices))) { stop("objects left unused in the directory") }
# Length insert and orientation checking.
if (!pseudo) {
keepers<-codes==0L | codes==2L
} else {
keepers <- !logical(length(codes))
}
valid.len <- frag.lens[keepers]
valid.insert <- inserts[keepers]
valid.ori <- orientations[keepers]
o <- order(valid.len, valid.ori, valid.insert)
auxiliary <- getPairData(tmpdir, param)
o2 <- do.call(order, auxiliary)
if (!identical(valid.len[o], auxiliary$length[o2])) { stop("extracted fragment sizes don't match up") }
if (!identical(valid.insert[o], auxiliary$insert[o2])) { stop("extracted inserts don't match up") }
if (!identical(valid.ori[o], auxiliary$orientation[o2])) { stop("extracted orientations don't match up") }
curdex <- h5ls(tmpdir)
curdex <- curdex[curdex$otype=="H5I_DATASET",][1,]
returned <- h5read(tmpdir, file.path(curdex$group, curdex$name))
processed <- diffHic:::.getStats(returned, basename(curdex$group)==curdex$name, used.frags)
return(head(data.frame(anchor1.id=returned$anchor1.id, anchor2.id=returned$anchor2.id, length=processed$length,
orientation=processed$orientation, insert=processed$insert)))
}
####################################################################################################
# Initiating testing with something fairly benign.
set.seed(0)
fname<-file.path(dir, "out");
max.cuts<-c(chrA=20L, chrB=10L, chrC=5L)
comp(fname, npairs=20, max.cuts=max.cuts)
comp(fname, npairs=50, max.cuts=max.cuts)
comp(fname, npairs=100, max.cuts=max.cuts)
# Ramping up the aggression in terms of self-circles.
comp(fname, npairs=20, max.cuts=c(chrA=3L))
comp(fname, npairs=50, max.cuts=c(chrA=3L))
comp(fname, npairs=100, max.cuts=c(chrA=3L))
# Increasing the number of reads all round.
comp(fname, npairs=200, size=c(500, 1000), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(500, 1000), max.cuts=max.cuts);
# Adding some singletons.
comp(fname, npairs=200, size=c(20, 100), max.cuts=max.cuts, singles=10)
comp(fname, npairs=200, size=c(20, 100), max.cuts=max.cuts, singles=50)
# Making the fragments smaller.
comp(fname, npairs=200, size=c(60, 100), rlen=50, max.cuts=max.cuts)
comp(fname, npairs=500, size=c(60, 100), rlen=50, max.cuts=max.cuts)
# Trying out negative spacings (i.e., non-filled genomes)
comp(fname, npairs=200, size=c(500, 1000), overhang=2, max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(500, 1000), overhang=2, max.cuts=max.cuts);
comp(fname, npairs=200, size=c(500, 1000), overhang=4, max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(500, 1000), overhang=4, max.cuts=max.cuts);
comp(fname, npairs=200, size=c(20, 100), overhang=2, max.cuts=max.cuts, singles=10)
comp(fname, npairs=200, size=c(20, 100), overhang=2, max.cuts=max.cuts, singles=50)
comp(fname, npairs=200, size=c(20, 100), overhang=4, max.cuts=max.cuts, singles=10)
comp(fname, npairs=200, size=c(20, 100), overhang=4, max.cuts=max.cuts, singles=50)
comp(fname, npairs=200, size=c(60, 100), overhang=4, rlen=50, max.cuts=max.cuts)
comp(fname, npairs=500, size=c(60, 100), overhang=4, rlen=50, max.cuts=max.cuts)
comp(fname, npairs=200, size=c(60, 100), overhang=2, rlen=50, max.cuts=max.cuts)
comp(fname, npairs=500, size=c(60, 100), overhang=2, rlen=50, max.cuts=max.cuts)
# Trying it out with some more elements in a more restricted space.
comp(fname, npairs=500, size=c(100, 500), max.cuts=c(chrA=2L))
comp(fname, npairs=200, size=c(500, 1000), max.cuts=c(chrA=2L, chrB=1L))
comp(fname, npairs=1000, size=c(20, 50), max.cuts=c(chrA=1L))
# Adding lots of chromosomes.
max.cuts<-c(chrA=5L, chrB=6L, chrC=7L, chrD=4L, chrE=3L, chrF=1L, chrG=2L)
comp(fname, npairs=200, size=c(500, 1000), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(50, 100), max.cuts=max.cuts);
comp(fname, npairs=200, size=c(100, 500), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(200, 300), max.cuts=max.cuts);
max.cuts<-c(chrD=5L, chrC=6L, chrA=7L, chrE=4L, chrG=3L, chrB=1L, chrF=2L) # Shuffled, to test effect of non-trivial 'm'.
comp(fname, npairs=200, size=c(500, 1000), max.cuts=max.cuts)
comp(fname, npairs=1000, size=c(50, 100), max.cuts=max.cuts);
comp(fname, npairs=200, size=c(100, 500), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(200, 300), max.cuts=max.cuts);
# Checking results with pseudo-ness
max.cuts<-c(chrA=20L, chrB=10L, chrC=5L)
comp(fname, npairs=20, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=50, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(200, 200))
comp(fname, npairs=1000, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(500, 500))
max.cuts<-c(chrB=20L, chrC=10L, chrA=5L) # Shuffled
comp(fname, npairs=20, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=50, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(200, 200))
comp(fname, npairs=1000, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(500, 500))
# Checking what happens when I add more or less chromosomes
max.cuts<-c(chrA=20L, chrB=10L)
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras="chrX") # should work
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras="chrX", pseudo=TRUE)
try({
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras=-1L) # fails due to removal of first chromosome.
})
try({
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras=-1L, pseudo=TRUE)
})
# Throwing more than 5000 read pairs in, to check that it still behaves past the 5000 read pair threshold for file dumping.
max.cuts<-c(chrA=20L, chrB=10L)
comp(fname, npairs=10000, max.cuts=max.cuts, sizes=c(50, 100))
comp(fname, npairs=10000, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), pseudo=TRUE)
comp(fname, npairs=10000, max.cuts=max.cuts, sizes=c(50, 100), storage=10) # Also checking that it does the same when we turn down the storage.
comp(fname, npairs=10000, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), pseudo=TRUE, storage=10)
###################################################################################################
# Trying to do simulations with chimeras is hellishly complicated, so we're just going to settle for
# consideration of chimeras with a fixed example.
hic.file<-system.file("exdata", "hic_sort.bam", package="diffHic")
break.file<-system.file("exdata", "cuts.rds", package="diffHic")
cuts<-readRDS(break.file)
param <- pairParam(cuts)
tmpdir<-file.path(dir, "gunk")
cntdir<-file.path(dir, "gunkcount")
# Setting up a function to name all read pairs based on their position, length and strand.
what <- c("qname", "rname", "pos", "qwidth", "strand")
first.read <- Rsamtools::scanBam(hic.file, param=ScanBamParam(flag=scanBamFlag(isPaired=TRUE, isFirstMateRead=TRUE, isSecondary=FALSE), what=what))[[1]]
second.read <- Rsamtools::scanBam(hic.file, param=ScanBamParam(flag=scanBamFlag(isPaired=TRUE, isFirstMateRead=FALSE, isSecondary=FALSE), what=what))[[1]]
fchar <- paste(first.read$rname, first.read$pos, first.read$strand, first.read$qwidth, sep=".")
schar <- paste(second.read$rname, second.read$pos, second.read$strand, second.read$qwidth, sep=".")
fqnames <- first.read$qname
sqnames <- second.read$qname
namefun <- function(extracted) {
xchar <- paste(seqnames(cuts[as.vector(extracted$anchor1.id)]), extracted$anchor1.pos,
ifelse(extracted$anchor1.len > 0, "+", "-"), abs(extracted$anchor1.len), sep=".")
ychar <- paste(seqnames(cuts[as.vector(extracted$anchor2.id)]), extracted$anchor2.pos,
ifelse(extracted$anchor2.len > 0, "+", "-"), abs(extracted$anchor2.len), sep=".")
output <- integer(nrow(extracted))
for (i in seq_len(nrow(extracted))) {
curx <- xchar[i]
cury <- ychar[i]
m <- (fchar %in% curx & schar %in% cury) | (fchar %in% cury & schar %in% curx)
stopifnot(sum(m)==1L)
output[i] <- which(m)
}
my.names <- fqnames[output]
stopifnot(identical(my.names, sqnames[output]))
return(my.names)
}
printfun<-function(dir) {
output<-list()
ix <- 1L
indices <- suppressWarnings(diffHic:::preloader(tmpdir))
for (ax in names(indices)) {
if (is.null(output[[ax]])) { output[[ax]]<-list() }
for (tx in names(indices[[ax]])) {
extracted <- h5read(dir, file.path(ax, tx))
processed <- diffHic:::.getStats(extracted, ax==tx, cuts)
output[[ax]][[tx]] <- data.frame(anchor1.id=extracted$anchor1.id, anchor2.id=extracted$anchor2.id,
length=processed$length, orientation=processed$orientation, insert=processed$insert)
rownames(output[[ax]][[tx]]) <- namefun(extracted)
ix <- ix + 1L
}
}
return(output)
}
# We also have 3 unmapped reads, 5 dangling ends, 2 self-circles, 2 singletons.
# For chimeras, all have mapped 5' and 3' ends, 7 of which are invalid.
preparePairs(hic.file, param, tmpdir, dedup=FALSE)
printfun(tmpdir)
# Alright, so once duplicates are removed, we lose:
# self.1 (--mapped/self.circles)
# other.1 (--mapped/other)
# dangling.1 (--mapped/dangling)
# chimeric.good.2 (--mapped/chimeric$mapped/multi)
# chimeric.invalid.1 (--mapped/chimeric$mapped/multi/invalid)
# chimeric.invalid.4 (--chimeric$multi/invalid)
# chimeric.good.4 (--mapped/chimeric$mapped/multi)
# chimeric.good.5 (--mapped/chimeric$mapped/multi)
#
# So, a loss of -7 to mapped, a loss of -1 to each of dangling and self.circles,
# a loss of -4 to chimeric$mapped, -5 to chimeric$multi and -2 to chimeric$invalid.
#
# Note that this will be considered the reference to which all downstream
# tests are compared, as dedup=TRUE is the default setting.
tmpdir2<-file.path(dir, "gunk2")
preparePairs(hic.file, param, tmpdir2)
printfun(tmpdir2)
# Once invalid chimeras are removed, we see a loss of all rows corresponding to
# invalid chimeras in printfun. No change in the statistics should be observed.
# Note that chimeric.invalid.4 is still okay as ther invalid component is removed
# by duplicate removal (for some reason; that shouldn't happen in real data).
preparePairs(hic.file, param, tmpdir2, ichim=FALSE)
printfun(tmpdir2)
# Throwing out those with poor mapping quality. We lose:
# good.1 (mapped -> filtered)
# good.3 (mapped -> filtered)
# dangling.2 (mapped/dangling -> filtered)
# dangling.3 (mapped/dangling -> filtered)
# chimeric.good.1 (--chimeric$multi)
# chimeric.invalid.2 (mapped/chimeric$mapped/multi/invalid -> filtered)
# chimeric.invalid.3 (--chimeric$multi/invalid)
# chimeric.invalid.6 (mapped/chimeric$mapped/multi/invalid -> filtered)
#
# So, a gain of +6 for filtered, a loss of -6 for mapped, a loss of -2
# for dangling, a loss of -2 for chimeric$mapped, -4 for chimeric$multi
# and -3 for chimeric$invalid. For printfun, we see:
# A/A = chimeric.invalid.5, good.2, chimeric.invalid.4, good.4
# B/A = good.8, good.5, chimeric.good.1, chimeric.invalid.3, chimeric.invalid.7
# B/B = good.7, good.6, chimeric.good.3, other.2
preparePairs(hic.file, param, tmpdir2, minq=100)
printfun(tmpdir2)
# Defining invalid chimeras based on distance instead of fragment ID. chimeric.good.1
# becomes an invalid chimera, as the distance between the 3' segment and the mate is
# 30 bp. This results in a +1 increase for invalid.chim.
preparePairs(hic.file, param, tmpdir2, chim.dist=20, ichim=FALSE)
printfun(tmpdir2)
# chimeric.invalid.6 now becomes a valid chimera, as each pair of 5' end and 3' mate end
# is now a proper pair (inward-facing and less than chim.dist). The same applies for
# chimeric.invalid.7. -2 for invalid.chim.
preparePairs(hic.file, param, tmpdir2, chim.dist=2000, ichim=FALSE)
printfun(tmpdir2)
###################################################################################################
# This tests what happens with chimeras where one of the segments is unmapped. This requires some
# care because unmapped reads don't get CIGAR strings, which makes diagnosing 5' behaviour difficult.
generator <- function(cig1, cig2, cig3, cig4) {
mapped <- "chrA 100"
unmapped <- "* 0"
out <- sprintf("@HD VN:1.3 SO:queryname
@SQ SN:chrA LN:200
x1 %i %s 200 %s * 0 0 NNNNN hhhhh
x1 %i %s 200 %s * 0 0 NNNNN hhhhh
x1 %i %s 200 %s * 0 0 NNNNN hhhhh
x1 %i %s 200 %s * 0 0 NNNNN hhhhh",
1+64 +ifelse(cig1=="*", 4, 0), ifelse(cig1!="*", mapped, unmapped), cig1,
1+64 +ifelse(cig2=="*", 4, 0)+256, ifelse(cig2!="*", mapped, unmapped), cig2,
1+128+ifelse(cig3=="*", 4, 0), ifelse(cig3!="*", mapped, unmapped), cig3,
1+128+ifelse(cig4=="*", 4, 0)+256, ifelse(cig4!="*", mapped, unmapped), cig4)
return(gsub(" +", "\t", out))
}
fout <- file.path(dir, "umap.sam")
# None of these should show up as a pair.
for (scenario in list(list("*", "*", "*", "*"),
list("5M5H", "*", "*", "*"),
list("*", "*", "5M5H", "*"),
list("*", "*", "*", "5H5M"),
list("*", "5H5M", "5M5H", "*"),
list("5M5H", "*", "*", "5H5M"))) {
writeLines(do.call(generator, scenario), con=fout)
sout <- Rsamtools::asBam(fout, file.path(dir, "umap"), overwrite=TRUE)
x <- preparePairs(sout, param=pairParam(GRanges("chrA", IRanges(c(1, 71), c(70, 200)))), file=file.path(dir, "whee.h5"))
stopifnot(x$pairs[["total"]]==1L)
stopifnot(x$pairs[["filtered"]]==1L)
stopifnot(x$chimeras[["total"]]==1L)
stopifnot(x$chimeras[["mapped"]]==0L)
}
# All of these should show up as a pair.
writeLines(generator("5M5H", "*", "5M5H", "*"), con=fout)
sout <- Rsamtools::asBam(fout, file.path(dir, "umap"), overwrite=TRUE)
x <- preparePairs(sout, param=pairParam(GRanges("chrA", IRanges(c(1, 71), c(70, 200)))), file=file.path(dir, "whee.h5"))
stopifnot(x$pairs[["total"]]==1L)
stopifnot(x$pairs[["mapped"]]==1L)
stopifnot(x$chimeras[["total"]]==1L)
stopifnot(x$chimeras[["mapped"]]==1L)
stopifnot(x$chimeras[["multi"]]==0L)
for (scenario in list(list("5M5H", "5H5M", "5M5H", "*"),
list("5M5H", "*", "5M5H", "5H5M"),
list("5M5H", "5H5M", "5M5H", "5H5M"))) {
writeLines(do.call(generator, scenario), con=fout)
sout <- Rsamtools::asBam(fout, file.path(dir, "umap"), overwrite=TRUE)
x <- preparePairs(sout, param=pairParam(GRanges("chrA", IRanges(c(1, 71), c(70, 200)))), file=file.path(dir, "whee.h5"))
stopifnot(x$pairs[["total"]]==1L)
stopifnot(x$pairs[["mapped"]]==1L)
stopifnot(x$chimeras[["total"]]==1L)
stopifnot(x$chimeras[["mapped"]]==1L)
stopifnot(x$chimeras[["multi"]]==1L)
}
###################################################################################################
unlink(dir, recursive=TRUE) # Cleaning up
###################################################################################################
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###################################################################################################
# This script is designed to test the pair-identifying capabilities of the hiC machinery i.e. preparePairs.
# We start with unit tests for individual components of the preparePairs C++ code.
suppressPackageStartupMessages(require(diffHic))
suppressPackageStartupMessages(require(GenomicAlignments))
source("simsam.R")
dir<-"hic-test"
dir.create(dir)
# Checking CIGAR.
checkCIGAR <- function(cigar, rstrand) {
output <- simsam(file.path(dir, "whee"), "chrA", 1, !rstrand, c("chrA"=1000),
cigar=cigar, len=cigarWidthAlongQuerySpace(cigar))
out <- .Call(diffHic:::cxx_test_parse_cigar, output)
if (is.character(out)) { stop(out) }
true.alen <- cigarWidthAlongReferenceSpace(cigar)
if (out[1]!=true.alen) { stop("mismatch in alignment length") }
offset <- 0
as.rle <- cigarToRleList(cigar)[[1]]
if (rstrand) { as.rle <- rev(as.rle) }
if (runValue(as.rle)[1]=="H") { offset <- runLength(as.rle)[1] }
if (out[2]!=offset) { stop("mismatch in offsets") }
return(c(alen=true.alen, offset=offset))
}
checkCIGAR("5H20M", TRUE)
checkCIGAR("5H20M", FALSE)
checkCIGAR("5H20M6H", TRUE)
checkCIGAR("5H20M6H", FALSE)
checkCIGAR("5H20M6S", TRUE)
checkCIGAR("5H20M6S", FALSE)
checkCIGAR("20M5I30M", TRUE)
checkCIGAR("20M5D30M", TRUE)
checkCIGAR("20M5N30M", TRUE)
checkCIGAR("20M5P30M", TRUE)
checkCIGAR("20M5X30M", TRUE)
checkCIGAR("20M5=30M", TRUE)
checkCIGAR("10M2I5M3D20M", TRUE)
checkCIGAR("10M2I3D5N20M", TRUE)
checkCIGAR("10M2P5M3D20I", TRUE)
checkCIGAR("10M2I3D5N20D", TRUE)
checkCIGAR("5H11M5D8I9X6=6H", TRUE)
checkCIGAR("5H20X5D8P9N72M", FALSE)
checkCIGAR("5S19M34=55D8X20M6S", TRUE)
checkCIGAR("5S1M3=5D18X2M6S", FALSE)
# Checking fragment assignment.
assign2fragment <- function(starts, ends, chr, pos, rstrand, len) {
out <- .Call(diffHic:::cxx_test_fragment_assign, starts, ends, chr, pos, rstrand, len)
if (is.character(out)) { stop(out) }
chr <- chr + 1L
if (rstrand) {
fiveprime <- min(pos + len -1L, tail(ends[[chr]], 1))
stopifnot(ends[[chr]][out] >= fiveprime && (out==1L || ends[[chr]][out-1] < fiveprime))
} else {
fiveprime <- pos
stopifnot(starts[[chr]][out] <= fiveprime && (out==length(starts[[chr]]) || starts[[chr]][out+1] > fiveprime))
}
out
}
starts <- list( c(1L, 100L, 200L, 300L, 400L, 500L), # chr1
c(1L, 100L, 200L, 300L, 400L, 500L)) # chr2
ends <- list( c(103L, 203L, 303L, 403L, 503L, 1000L), # chr1
c(103L, 203L, 303L, 403L, 503L, 1000L)) # chr2
assign2fragment(starts, ends, 0L, 94L, TRUE, 10L)
assign2fragment(starts, ends, 0L, 95L, TRUE, 10L)
assign2fragment(starts, ends, 0L, 99L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 100L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 203L, TRUE, 1L)
assign2fragment(starts, ends, 0L, 204L, TRUE, 1L)
assign2fragment(starts, ends, 0L, 209L, FALSE, 1L)
assign2fragment(starts, ends, 0L, 300L, FALSE, 1L)
assign2fragment(starts, ends, 1L, 1L, FALSE, 10L)
assign2fragment(starts, ends, 1L, 991L, FALSE, 10L)
assign2fragment(starts, ends, 1L, 992L, FALSE, 10L)
assign2fragment(starts, ends, 1L, 991L, TRUE, 10L)
assign2fragment(starts, ends, 1L, 992L, TRUE, 10L)
starts <- list( c(1L, 1L, 100L, 200L, 300L, 400L, 500L, 997L), # chr1, with nesting at the start and end.
c(1L, 1L, 100L, 200L, 300L, 400L, 500L, 997L)) # chr2
ends <- list( c(4L, 103L, 203L, 303L, 403L, 503L, 1000L, 1000L), # chr1
c(4L, 103L, 203L, 303L, 403L, 503L, 1000L, 1000L)) # chr2
assign2fragment(starts, ends, 0L, 1L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 1L, TRUE, 10L)
assign2fragment(starts, ends, 0L, 991L, FALSE, 10L)
assign2fragment(starts, ends, 0L, 991L, TRUE, 10L)
try(assign2fragment(starts, ends, 0L, 1000L, TRUE, 10L)) # This should fail, as it gets assigned into the nested fragment.
###################################################################################################
# We also set up a full simulation for the entire function.
suppressPackageStartupMessages(require("rhdf5"))
comp <- function (fname, npairs, max.cuts, sizes=c(100, 500), singles=0, rlen=10, overhang=0L, pseudo=FALSE, extras=NULL, storage=5000) {
################ SETTING UP THE FRAGMENTS ###################
rlen <- as.integer(rlen)
overhang <- as.integer(overhang)
if (min(sizes) <= rlen) { stop("min fragment must be greater than read length") }
# Necessary for proper assignment, especially at the start of the chromosome when reverse
# reads are bounded at zero (i.e. their 5' ends would not be defined if 1+rlen > fragmentsize)
# Randomly generating fragment lengths for the chromosome.
fragments <- cut.starts <- outfrags <- list()
chromosomes <- integer(length(max.cuts))
for (i in seq_along(max.cuts)) {
fragments[[i]] <- as.integer(round(runif(max.cuts[[i]], sizes[1], sizes[2])))
ends <- cumsum(fragments[[i]]) - seq_len(max.cuts[[i]])*overhang
if (max.cuts[[i]] > 1L) {
cut.starts[[i]] <- c(1L, (ends+1L)[seq_len(max.cuts[[i]]-1)])
} else {
cut.starts[[i]] <- 1L
}
outfrags[[i]] <- GRanges(names(max.cuts)[i], IRanges(cut.starts[[i]], ends+overhang))
chromosomes[i] <- tail(ends, 1)+overhang
}
seq.names <- names(max.cuts)
seq.lengths <- chromosomes
suppressWarnings(outfrags <- do.call(c, outfrags))
seqlevels(outfrags) <- seq.names
seqlengths(outfrags) <- setNames(seq.lengths, seq.names)
names(chromosomes) <- names(max.cuts)
names(fragments) <- names(max.cuts)
names(cut.starts) <- names(max.cuts)
################ SETTING UP THE READ PAIRS ###################
# Randomly generating reads (a la getPESizes' example).
names <- paste('x', rep(seq_len(npairs), 2), sep=".")
chrs <- sample(length(chromosomes), length(names), replace=TRUE)
pos <- integer(length(names));
frag.ids <- integer(length(names))
str <- rbinom(length(names), 1, 0.5)==1
# Assigning positions to all of them. Some finesse is necessary when
# there is no overhang (i.e. original genome with positive overhang). We
# still allow reads to span restriction sites, though.
for (i in seq_along(chromosomes)) {
current <- chrs==i
chosen.frags <- as.integer(runif(sum(current), 1, length(fragments[[i]])+1))
frag.ids[current] <- chosen.frags
my.ends <- cut.starts[[i]] + fragments[[i]]
# Note that as.integer(runif(1, a, b)) samples from [a, b), as runif() will never actually generate 'b'.
# So, this will only generate positions after and including the start position for the chosen fragment,
# but before and not including the start position for the next fragment (or 1-past the end of the chromosome).
cur.for <- str[current]
forward.frag <- chosen.frags[cur.for]
forward.min <- cut.starts[[i]][forward.frag]
forward.max <- ifelse(forward.frag!=length(cut.starts[[i]]), cut.starts[[i]][forward.frag+1L], my.ends[forward.frag])
pos[current][cur.for] <- as.integer(runif(sum(cur.for), forward.min, forward.max))
# Recall that the 'ends' are 1-past the last base of the fragment, so this runif() will sample from
# the first non-overlapping base of the current fragment to the last base of the current fragment
# (it's [a, b), so when b is 1-past the last base, sampling will include the last base).
reverse.frag <- chosen.frags[!cur.for]
reverse.min <- integer(sum(!cur.for))
possible.zero <- reverse.frag==1L
reverse.min[possible.zero] <- 1L
reverse.min[!possible.zero] <- my.ends[reverse.frag[!possible.zero]-1L]
reverse.max <- my.ends[reverse.frag]
pos[current][!cur.for] <- pmax(1L, as.integer(runif(sum(!cur.for), reverse.min, reverse.max)) - rlen + 1L)
}
# Throwing them into the SAM file generator. Note that chromosome names are ordered inside.
# If this differs from the order in 'max.cuts', it will test the ability of preparePairs to match them up correctly.
reversi <- c(seq_len(npairs) + npairs, seq_len(npairs))
out <- simsam(fname, names(chromosomes)[chrs], pos, str, chromosomes, names=names, len=rlen,
is.first=rep(c(TRUE, FALSE), each=npairs), is.paired=TRUE,
mate.chr=names(chromosomes)[chrs][reversi], mate.pos=pos[reversi], mate.str=str[reversi])
if (singles) {
# Adding some singles. You'll get some warnings regarding overhang regions as we're not finessing it.
snames <- schrs <- spos <- NULL
snames <- paste('y', seq_len(singles), sep=".")
schrs <- sample(length(chromosomes), singles, replace=TRUE)
spos <- integer(singles);
for (i in seq_along(chromosomes)) {
scurrent <- schrs==i;
spos[scurrent] <- as.integer(round(runif(sum(scurrent), 1, chromosomes[i])))
}
tempname <- file.path(dir, "temp")
sstr <- rbinom(singles, 1, 0.5)==1
out2 <- simsam(tempname, names(chromosomes)[schrs], spos, sstr, chromosomes, names=snames, len=rlen)
more.temp <- file.path(dir, "temp2")
out <- mergeBam(c(out, out2), more.temp, indexDestination=TRUE, overwrite=TRUE)
file.rename(more.temp, out)
}
# Resorting by name.
temp <- sortBam(out, "temp", byQname=TRUE)
file.rename(temp, out)
################ THEORETICAL MATCH ###################
# This gets the status: 0 for okay, 1 for dangling end, 3 for self.circles and and 2 for other stuff.
getstatus <- function(chr1, pos1, frag1, str1, chr2, pos2, frag2, str2) {
codes <- integer(length(chr1))
potentials <- chr1==chr2 & frag1==frag2
same.str <- str1==str2;
codes[ same.str & potentials ] <- 2L
potentials <- potentials & !same.str
self.circle <- (str1 & pos2+rlen <= pos1) | (str2 & pos1+rlen <= pos2);
codes[ self.circle & potentials ] <- 3L
potentials <- potentials & !self.circle
# Not worrying about overextension; these are now considered to be dangling ends,
# as sequencing/trimming/alignment errors can often result in a few overextended bases.
# overextension <- (str1 & pos1 > pos2) | (str2 & pos2 > pos1) # Don't need to check +rlen, as they're al the same.
# codes[ overextension & potentials ] <- 2L
# potentials <- potentials & !overextension
codes[potentials] <- 1L
return(codes)
}
# This gets the distance from the 5' end of the read to the next restriction site that it is pointing to (past the one it covers, if it is incomplete).
getlen <- function(chr, pos, start, str) {
len.out <- integer(length(chr))
for (x in seq_along(cut.starts)) {
chosen <- x==chr
dist2cut <- ifelse(str[chosen], cut.starts[[x]][start[chosen]]+fragments[[x]][start[chosen]]-pos[chosen],
pos[chosen]+rlen-cut.starts[[x]][start[chosen]])
len.out[chosen] <- dist2cut
}
return(len.out)
}
# Now, actually assembling the theoretical values.
primary <- seq_len(npairs)
secondary<- npairs + primary
pchrs <- chrs[primary]
ppos <- pos[primary]
pfrag <- frag.ids[primary]
pstr <- str[primary]
schrs <- chrs[secondary]
spos <- pos[secondary]
sfrag <- frag.ids[secondary]
sstr <- str[secondary]
codes <- getstatus(pchrs, ppos, pfrag, pstr, schrs, spos, sfrag, sstr)
if (pseudo) {
frag.lens <- rep(NA_integer_, npairs)
pfrag[] <- 0L
sfrag[] <- 0L
} else {
frag.lens <- getlen(pchrs, ppos, pfrag, pstr) + getlen(schrs, spos, sfrag, sstr)
}
ppos5 <- ifelse(pstr, ppos, ppos+rlen -1L)
spos5 <- ifelse(sstr, spos, spos+rlen -1L)
panchor <- pchrs > schrs | (pchrs==schrs & pfrag > sfrag) | (pchrs==schrs & pfrag==sfrag & ppos5 > spos5)
inserts <- ifelse(pchrs==schrs, pmax(ppos, spos)-pmin(spos, ppos)+rlen, NA)
orientations <- ifelse(pstr, 0L, ifelse(panchor, 1L, 2L))+ifelse(sstr, 0L, ifelse(panchor, 2L, 1L))
################ ACTUAL MATCH ###################
# Assembling the output list for comparison.
# Adding an extra chromosome to the start, to see if the match is still correct when it's not 1:1.
# Also deleting a chromosome to trigger an error.
if (!is.null(extras)) {
if (extras[1]==-1L) {
used.frags <- dropSeqlevels(outfrags, seqnames(outfrags)[1], pruning.mode="coarse")
} else {
used.frags <- suppressWarnings(c(GRanges(extras, IRanges(1, 100)), outfrags))
}
} else {
used.frags <- outfrags
}
tmpdir<-paste0(fname, "_temp")
param <- pairParam(fragments=used.frags)
if (pseudo) {
# Special behaviour; faster assignment into bins, no removal of dangling ends/self-cirlces
# (as these concepts are meaningless for arbitrary bins).
param <- pairParam(used.frags[0])
diagnostics <- preparePairs(out, param, tmpdir, output.dir=file.path(dir, "whee"), storage=storage)
} else {
diagnostics <- preparePairs(out, param, tmpdir, output.dir=file.path(dir, "whee"), storage=storage)
stopifnot(sum(codes==1L)==diagnostics$same.id[["dangling"]])
stopifnot(sum(codes==3L)==diagnostics$same.id[["self.circle"]])
stopifnot(length(codes)==diagnostics$pairs[["total"]])
stopifnot(singles==diagnostics$singles)
# No support for testing chimeras, we use a fixed example below.
stopifnot(diagnostics$unmapped.chimeras==0L)
stopifnot(diagnostics$chimeras[["total"]]==0L)
stopifnot(diagnostics$chimeras[["mapped"]]==0L)
stopifnot(diagnostics$chimeras[["invalid"]]==0L)
}
# Anchor1/anchor2 synchronisation is determined by order in 'fragments' (and thusly, in max.cuts).
if (pseudo) {
offset <- integer(length(chromosomes)) # 'extras' don't matter, as ID is set to zero anyway.
} else {
offset <- c(0L, cumsum(max.cuts))
names(offset) <- NULL
offset <- offset + length(extras)
}
indices <- diffHic:::preloader(tmpdir)
fchrs <- as.character(seqnames(used.frags))
for (i in seq_along(max.cuts)) {
for (j in seq_len(i)) {
# Getting the fragment IDs for the remaining pairs for this chromosome.
stuff <- (pchrs==i & schrs==j) | (pchrs==j & schrs==i)
if (!pseudo) { stuff <- stuff & (codes==0L | codes==2L) }
pids <- pfrag[stuff]
sids <- sfrag[stuff]
pps <- ppos[stuff]
sps <- spos[stuff]
pls <- ifelse(pstr[stuff], 1L, -1L)*rlen
sls <- ifelse(sstr[stuff], 1L, -1L)*rlen
which.is.which <- panchor[stuff]
anchor1 <- ifelse(which.is.which, pids, sids) + offset[i]
anchor2 <- ifelse(which.is.which, sids, pids) + offset[j]
ap1 <- ifelse(which.is.which, pps, sps)
ap2 <- ifelse(which.is.which, sps, pps)
al1 <- ifelse(which.is.which, pls, sls)
al2 <- ifelse(which.is.which, sls, pls)
totes <- frag.lens[stuff]
cur.ori <- orientations[stuff]
cur.insert <- inserts[stuff]
o <- order(anchor1, anchor2, ap1, ap2, al1, al2, totes, cur.ori, cur.insert)
# Extracting data from HDF5 and computing statistics.
achr <- names(max.cuts)[i]
tchr <- names(max.cuts)[j]
if (!(achr%in%names(indices)) || !(tchr %in% names(indices[[achr]]))) {
if (length(o)) { stop("true interactions are missing") }
next
}
current <- h5read(tmpdir, file.path(achr, tchr))
for (x in seq_len(ncol(current))) { attributes(current[,x]) <- NULL }
collated <- diffHic:::.getStats(current, achr==tchr, used.frags)
# Checking anchor1/anchor2/length/orientation/insert statistics (sorting on everything to ensure comparability).
o2 <- order(current$anchor1.id, current$anchor2.id, current$anchor1.pos, current$anchor2.pos,
current$anchor1.len, current$anchor2.len, collated$length, collated$orientation, collated$insert)
stopifnot(identical(current$anchor1.id[o2], anchor1[o]))
stopifnot(identical(current$anchor2.id[o2], anchor2[o]))
stopifnot(identical(current$anchor1.pos[o2], ap1[o]))
stopifnot(identical(current$anchor2.pos[o2], ap2[o]))
stopifnot(identical(current$anchor1.len[o2], al1[o]))
stopifnot(identical(current$anchor2.len[o2], al2[o]))
stopifnot(identical(collated$length[o2], totes[o]))
stopifnot(identical(collated$orientation[o2], cur.ori[o]))
stopifnot(identical(collated$insert[o2], cur.insert[o]))
# Checking that choice and ordering of anchor1 and anchor2 is sane.
current5.1 <- current$anchor1.pos - pmin(0L, current$anchor1.len + 1L)
current5.2 <- current$anchor2.pos - pmin(0L, current$anchor2.len + 1L)
if (!pseudo) {
stopifnot(all(current$anchor1.id >= current$anchor2.id))
stopifnot(all((current5.1 >= current5.2)[current$anchor1.id==current$anchor2.id]))
o <- order(current$anchor1.id, current$anchor2.id)
stopifnot(!is.unsorted(o))
} else {
stopifnot(all(current$anchor1.id==0L & current$anchor2.id==0L))
if (i==j) {
if (!all(current5.1 >= current5.2)) {
print(current)
}
stopifnot(all(current5.1 >= current5.2))
}
}
# Checking that we're looking at the right combination.
if (!pseudo) {
uniq.a <- unique(fchrs[current$anchor1.id])
uniq.t <- unique(fchrs[current$anchor2.id])
if (length(uniq.a)!=1L || length(uniq.t)!=1L) { stop("file contains more than one combination") }
if (achr!=uniq.a || tchr!=uniq.t) { stop("file contains the incorrect combination") }
}
# Wiping it out to indicate that we've visited this place.
indices[[achr]][[tchr]]<-NULL
}
}
# Checking there's nothing left.
if (!is.null(unlist(indices))) { stop("objects left unused in the directory") }
# Length insert and orientation checking.
if (!pseudo) {
keepers<-codes==0L | codes==2L
} else {
keepers <- !logical(length(codes))
}
valid.len <- frag.lens[keepers]
valid.insert <- inserts[keepers]
valid.ori <- orientations[keepers]
o <- order(valid.len, valid.ori, valid.insert)
auxiliary <- getPairData(tmpdir, param)
o2 <- do.call(order, auxiliary)
if (!identical(valid.len[o], auxiliary$length[o2])) { stop("extracted fragment sizes don't match up") }
if (!identical(valid.insert[o], auxiliary$insert[o2])) { stop("extracted inserts don't match up") }
if (!identical(valid.ori[o], auxiliary$orientation[o2])) { stop("extracted orientations don't match up") }
curdex <- h5ls(tmpdir)
curdex <- curdex[curdex$otype=="H5I_DATASET",][1,]
returned <- h5read(tmpdir, file.path(curdex$group, curdex$name))
processed <- diffHic:::.getStats(returned, basename(curdex$group)==curdex$name, used.frags)
return(head(data.frame(anchor1.id=returned$anchor1.id, anchor2.id=returned$anchor2.id, length=processed$length,
orientation=processed$orientation, insert=processed$insert)))
}
####################################################################################################
# Initiating testing with something fairly benign.
set.seed(0)
fname<-file.path(dir, "out");
max.cuts<-c(chrA=20L, chrB=10L, chrC=5L)
comp(fname, npairs=20, max.cuts=max.cuts)
comp(fname, npairs=50, max.cuts=max.cuts)
comp(fname, npairs=100, max.cuts=max.cuts)
# Ramping up the aggression in terms of self-circles.
comp(fname, npairs=20, max.cuts=c(chrA=3L))
comp(fname, npairs=50, max.cuts=c(chrA=3L))
comp(fname, npairs=100, max.cuts=c(chrA=3L))
# Increasing the number of reads all round.
comp(fname, npairs=200, size=c(500, 1000), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(500, 1000), max.cuts=max.cuts);
# Adding some singletons.
comp(fname, npairs=200, size=c(20, 100), max.cuts=max.cuts, singles=10)
comp(fname, npairs=200, size=c(20, 100), max.cuts=max.cuts, singles=50)
# Making the fragments smaller.
comp(fname, npairs=200, size=c(60, 100), rlen=50, max.cuts=max.cuts)
comp(fname, npairs=500, size=c(60, 100), rlen=50, max.cuts=max.cuts)
# Trying out negative spacings (i.e., non-filled genomes)
comp(fname, npairs=200, size=c(500, 1000), overhang=2, max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(500, 1000), overhang=2, max.cuts=max.cuts);
comp(fname, npairs=200, size=c(500, 1000), overhang=4, max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(500, 1000), overhang=4, max.cuts=max.cuts);
comp(fname, npairs=200, size=c(20, 100), overhang=2, max.cuts=max.cuts, singles=10)
comp(fname, npairs=200, size=c(20, 100), overhang=2, max.cuts=max.cuts, singles=50)
comp(fname, npairs=200, size=c(20, 100), overhang=4, max.cuts=max.cuts, singles=10)
comp(fname, npairs=200, size=c(20, 100), overhang=4, max.cuts=max.cuts, singles=50)
comp(fname, npairs=200, size=c(60, 100), overhang=4, rlen=50, max.cuts=max.cuts)
comp(fname, npairs=500, size=c(60, 100), overhang=4, rlen=50, max.cuts=max.cuts)
comp(fname, npairs=200, size=c(60, 100), overhang=2, rlen=50, max.cuts=max.cuts)
comp(fname, npairs=500, size=c(60, 100), overhang=2, rlen=50, max.cuts=max.cuts)
# Trying it out with some more elements in a more restricted space.
comp(fname, npairs=500, size=c(100, 500), max.cuts=c(chrA=2L))
comp(fname, npairs=200, size=c(500, 1000), max.cuts=c(chrA=2L, chrB=1L))
comp(fname, npairs=1000, size=c(20, 50), max.cuts=c(chrA=1L))
# Adding lots of chromosomes.
max.cuts<-c(chrA=5L, chrB=6L, chrC=7L, chrD=4L, chrE=3L, chrF=1L, chrG=2L)
comp(fname, npairs=200, size=c(500, 1000), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(50, 100), max.cuts=max.cuts);
comp(fname, npairs=200, size=c(100, 500), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(200, 300), max.cuts=max.cuts);
max.cuts<-c(chrD=5L, chrC=6L, chrA=7L, chrE=4L, chrG=3L, chrB=1L, chrF=2L) # Shuffled, to test effect of non-trivial 'm'.
comp(fname, npairs=200, size=c(500, 1000), max.cuts=max.cuts)
comp(fname, npairs=1000, size=c(50, 100), max.cuts=max.cuts);
comp(fname, npairs=200, size=c(100, 500), max.cuts=max.cuts);
comp(fname, npairs=1000, size=c(200, 300), max.cuts=max.cuts);
# Checking results with pseudo-ness
max.cuts<-c(chrA=20L, chrB=10L, chrC=5L)
comp(fname, npairs=20, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=50, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(200, 200))
comp(fname, npairs=1000, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(500, 500))
max.cuts<-c(chrB=20L, chrC=10L, chrA=5L) # Shuffled
comp(fname, npairs=20, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=50, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(100, 100))
comp(fname, npairs=100, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(200, 200))
comp(fname, npairs=1000, max.cuts=max.cuts, pseudo=TRUE, overhang=0, sizes=c(500, 500))
# Checking what happens when I add more or less chromosomes
max.cuts<-c(chrA=20L, chrB=10L)
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras="chrX") # should work
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras="chrX", pseudo=TRUE)
try({
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras=-1L) # fails due to removal of first chromosome.
})
try({
comp(fname, npairs=20, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), extras=-1L, pseudo=TRUE)
})
# Throwing more than 5000 read pairs in, to check that it still behaves past the 5000 read pair threshold for file dumping.
max.cuts<-c(chrA=20L, chrB=10L)
comp(fname, npairs=10000, max.cuts=max.cuts, sizes=c(50, 100))
comp(fname, npairs=10000, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), pseudo=TRUE)
comp(fname, npairs=10000, max.cuts=max.cuts, sizes=c(50, 100), storage=10) # Also checking that it does the same when we turn down the storage.
comp(fname, npairs=10000, max.cuts=max.cuts, overhang=0, sizes=c(100, 100), pseudo=TRUE, storage=10)
###################################################################################################
# Trying to do simulations with chimeras is hellishly complicated, so we're just going to settle for
# consideration of chimeras with a fixed example.
hic.file<-system.file("exdata", "hic_sort.bam", package="diffHic")
break.file<-system.file("exdata", "cuts.rds", package="diffHic")
cuts<-readRDS(break.file)
param <- pairParam(cuts)
tmpdir<-file.path(dir, "gunk")
cntdir<-file.path(dir, "gunkcount")
# Setting up a function to name all read pairs based on their position, length and strand.
what <- c("qname", "rname", "pos", "qwidth", "strand")
first.read <- Rsamtools::scanBam(hic.file, param=ScanBamParam(flag=scanBamFlag(isPaired=TRUE, isFirstMateRead=TRUE, isSecondary=FALSE), what=what))[[1]]
second.read <- Rsamtools::scanBam(hic.file, param=ScanBamParam(flag=scanBamFlag(isPaired=TRUE, isFirstMateRead=FALSE, isSecondary=FALSE), what=what))[[1]]
fchar <- paste(first.read$rname, first.read$pos, first.read$strand, first.read$qwidth, sep=".")
schar <- paste(second.read$rname, second.read$pos, second.read$strand, second.read$qwidth, sep=".")
fqnames <- first.read$qname
sqnames <- second.read$qname
namefun <- function(extracted) {
xchar <- paste(seqnames(cuts[as.vector(extracted$anchor1.id)]), extracted$anchor1.pos,
ifelse(extracted$anchor1.len > 0, "+", "-"), abs(extracted$anchor1.len), sep=".")
ychar <- paste(seqnames(cuts[as.vector(extracted$anchor2.id)]), extracted$anchor2.pos,
ifelse(extracted$anchor2.len > 0, "+", "-"), abs(extracted$anchor2.len), sep=".")
output <- integer(nrow(extracted))
for (i in seq_len(nrow(extracted))) {
curx <- xchar[i]
cury <- ychar[i]
m <- (fchar %in% curx & schar %in% cury) | (fchar %in% cury & schar %in% curx)
stopifnot(sum(m)==1L)
output[i] <- which(m)
}
my.names <- fqnames[output]
stopifnot(identical(my.names, sqnames[output]))
return(my.names)
}
printfun<-function(dir) {
output<-list()
ix <- 1L
indices <- suppressWarnings(diffHic:::preloader(tmpdir))
for (ax in names(indices)) {
if (is.null(output[[ax]])) { output[[ax]]<-list() }
for (tx in names(indices[[ax]])) {
extracted <- h5read(dir, file.path(ax, tx))
processed <- diffHic:::.getStats(extracted, ax==tx, cuts)
output[[ax]][[tx]] <- data.frame(anchor1.id=extracted$anchor1.id, anchor2.id=extracted$anchor2.id,
length=processed$length, orientation=processed$orientation, insert=processed$insert)
rownames(output[[ax]][[tx]]) <- namefun(extracted)
ix <- ix + 1L
}
}
return(output)
}
# We also have 3 unmapped reads, 5 dangling ends, 2 self-circles, 2 singletons.
# For chimeras, all have mapped 5' and 3' ends, 7 of which are invalid.
preparePairs(hic.file, param, tmpdir, dedup=FALSE)
printfun(tmpdir)
# Alright, so once duplicates are removed, we lose:
# self.1 (--mapped/self.circles)
# other.1 (--mapped/other)
# dangling.1 (--mapped/dangling)
# chimeric.good.2 (--mapped/chimeric$mapped/multi)
# chimeric.invalid.1 (--mapped/chimeric$mapped/multi/invalid)
# chimeric.invalid.4 (--chimeric$multi/invalid)
# chimeric.good.4 (--mapped/chimeric$mapped/multi)
# chimeric.good.5 (--mapped/chimeric$mapped/multi)
#
# So, a loss of -7 to mapped, a loss of -1 to each of dangling and self.circles,
# a loss of -4 to chimeric$mapped, -5 to chimeric$multi and -2 to chimeric$invalid.
#
# Note that this will be considered the reference to which all downstream
# tests are compared, as dedup=TRUE is the default setting.
tmpdir2<-file.path(dir, "gunk2")
preparePairs(hic.file, param, tmpdir2)
printfun(tmpdir2)
# Once invalid chimeras are removed, we see a loss of all rows corresponding to
# invalid chimeras in printfun. No change in the statistics should be observed.
# Note that chimeric.invalid.4 is still okay as ther invalid component is removed
# by duplicate removal (for some reason; that shouldn't happen in real data).
preparePairs(hic.file, param, tmpdir2, ichim=FALSE)
printfun(tmpdir2)
# Throwing out those with poor mapping quality. We lose:
# good.1 (mapped -> filtered)
# good.3 (mapped -> filtered)
# dangling.2 (mapped/dangling -> filtered)
# dangling.3 (mapped/dangling -> filtered)
# chimeric.good.1 (--chimeric$multi)
# chimeric.invalid.2 (mapped/chimeric$mapped/multi/invalid -> filtered)
# chimeric.invalid.3 (--chimeric$multi/invalid)
# chimeric.invalid.6 (mapped/chimeric$mapped/multi/invalid -> filtered)
#
# So, a gain of +6 for filtered, a loss of -6 for mapped, a loss of -2
# for dangling, a loss of -2 for chimeric$mapped, -4 for chimeric$multi
# and -3 for chimeric$invalid. For printfun, we see:
# A/A = chimeric.invalid.5, good.2, chimeric.invalid.4, good.4
# B/A = good.8, good.5, chimeric.good.1, chimeric.invalid.3, chimeric.invalid.7
# B/B = good.7, good.6, chimeric.good.3, other.2
preparePairs(hic.file, param, tmpdir2, minq=100)
printfun(tmpdir2)
# Defining invalid chimeras based on distance instead of fragment ID. chimeric.good.1
# becomes an invalid chimera, as the distance between the 3' segment and the mate is
# 30 bp. This results in a +1 increase for invalid.chim.
preparePairs(hic.file, param, tmpdir2, chim.dist=20, ichim=FALSE)
printfun(tmpdir2)
# chimeric.invalid.6 now becomes a valid chimera, as each pair of 5' end and 3' mate end
# is now a proper pair (inward-facing and less than chim.dist). The same applies for
# chimeric.invalid.7. -2 for invalid.chim.
preparePairs(hic.file, param, tmpdir2, chim.dist=2000, ichim=FALSE)
printfun(tmpdir2)
###################################################################################################
# This tests what happens with chimeras where one of the segments is unmapped. This requires some
# care because unmapped reads don't get CIGAR strings, which makes diagnosing 5' behaviour difficult.
generator <- function(cig1, cig2, cig3, cig4) {
mapped <- "chrA 100"
unmapped <- "* 0"
out <- sprintf("@HD VN:1.3 SO:queryname
@SQ SN:chrA LN:200
x1 %i %s 200 %s * 0 0 NNNNN hhhhh
x1 %i %s 200 %s * 0 0 NNNNN hhhhh
x1 %i %s 200 %s * 0 0 NNNNN hhhhh
x1 %i %s 200 %s * 0 0 NNNNN hhhhh",
1+64 +ifelse(cig1=="*", 4, 0), ifelse(cig1!="*", mapped, unmapped), cig1,
1+64 +ifelse(cig2=="*", 4, 0)+256, ifelse(cig2!="*", mapped, unmapped), cig2,
1+128+ifelse(cig3=="*", 4, 0), ifelse(cig3!="*", mapped, unmapped), cig3,
1+128+ifelse(cig4=="*", 4, 0)+256, ifelse(cig4!="*", mapped, unmapped), cig4)
return(gsub(" +", "\t", out))
}
fout <- file.path(dir, "umap.sam")
# None of these should show up as a pair.
for (scenario in list(list("*", "*", "*", "*"),
list("5M5H", "*", "*", "*"),
list("*", "*", "5M5H", "*"),
list("*", "*", "*", "5H5M"),
list("*", "5H5M", "5M5H", "*"),
list("5M5H", "*", "*", "5H5M"))) {
writeLines(do.call(generator, scenario), con=fout)
sout <- Rsamtools::asBam(fout, file.path(dir, "umap"), overwrite=TRUE)
x <- preparePairs(sout, param=pairParam(GRanges("chrA", IRanges(c(1, 71), c(70, 200)))), file=file.path(dir, "whee.h5"))
stopifnot(x$pairs[["total"]]==1L)
stopifnot(x$pairs[["filtered"]]==1L)
stopifnot(x$chimeras[["total"]]==1L)
stopifnot(x$chimeras[["mapped"]]==0L)
}
# All of these should show up as a pair.
writeLines(generator("5M5H", "*", "5M5H", "*"), con=fout)
sout <- Rsamtools::asBam(fout, file.path(dir, "umap"), overwrite=TRUE)
x <- preparePairs(sout, param=pairParam(GRanges("chrA", IRanges(c(1, 71), c(70, 200)))), file=file.path(dir, "whee.h5"))
stopifnot(x$pairs[["total"]]==1L)
stopifnot(x$pairs[["mapped"]]==1L)
stopifnot(x$chimeras[["total"]]==1L)
stopifnot(x$chimeras[["mapped"]]==1L)
stopifnot(x$chimeras[["multi"]]==0L)
for (scenario in list(list("5M5H", "5H5M", "5M5H", "*"),
list("5M5H", "*", "5M5H", "5H5M"),
list("5M5H", "5H5M", "5M5H", "5H5M"))) {
writeLines(do.call(generator, scenario), con=fout)
sout <- Rsamtools::asBam(fout, file.path(dir, "umap"), overwrite=TRUE)
x <- preparePairs(sout, param=pairParam(GRanges("chrA", IRanges(c(1, 71), c(70, 200)))), file=file.path(dir, "whee.h5"))
stopifnot(x$pairs[["total"]]==1L)
stopifnot(x$pairs[["mapped"]]==1L)
stopifnot(x$chimeras[["total"]]==1L)
stopifnot(x$chimeras[["mapped"]]==1L)
stopifnot(x$chimeras[["multi"]]==1L)
}
###################################################################################################
unlink(dir, recursive=TRUE) # Cleaning up
###################################################################################################
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