Additions for COVID-19 project: Abundance of multiple pathogenic and …

…host amplicons

3_combine.pl

@@ -106,8 +106,13 @@
 print "[3] Done getting junction counts\n";
 
 
+# Generate files for expression analysis
+system "$path/R/combine_reads_expression.R -e $evTab -t $treatTab -d fwd -c $cores $outDir" and
+    die "[3] Error when combining reads for expression\n";
+print "[3] Done generating tables for expression\n";
+
+
 # Get PSI, RPM, pseudo counts etc. per well
-mkdir $outDir."/welldata" unless (-e $outDir."/welldata");
 system "$path/R/compute_psi_rpm.R -c $cores $outDir -e $evTab" and
     die "[3] Error when calculating PSI etc.\n";
 print "[3] Done computing PSI\n";

R/combine_reads_expression.R

@@ -0,0 +1,230 @@
+#!/usr/bin/env Rscript
+### Combine read counts and RPM from raw counts per well for SARS-CoV-2 project
+### U. Braunschweig 2020
+
+
+libMissing <- !require(optparse, quietly=T)
+if (libMissing) {stop("Failed to load R package 'optparse'")}
+
+#### Options
+args <- commandArgs(TRUE)
+
+option.list <- list(
+    make_option(c("-e", "--eventTab"), type="character",
+        help="CSV file describing the amplicons - see README"),
+    make_option(c("-t", "--treatTab"), type="character",
+        help="CSV file describing the samples - see README"),
+    make_option(c("-d", "--direction"),  default="both",
+                help="Which reads to use. 'fwd' for forward/R1, 'rev' for reverse/R1, or 'both for mean [%default]"),
+    make_option(c("-i", "--imbal"),  default=1.5,
+                help="Maximum imbalance between fwd and rev reads before a warning is printed [%default]"),
+    make_option(c("-b", "--imbalCts"),  default=20,
+                help="Minimum reads (average of fwd and rev) to do imbalance test [%default]"),
+    make_option(c("-c", "--cores"),  default=1,
+                help="Number of CPUs [%default]")
+
+)
+parser <- OptionParser(option_list=option.list,
+                       usage="usage: %prog [options] OUTDIR",
+                       description="Combine raw reads from invidual samples for expression analysis")
+opt <- parse_args(parser, args=args, positional_arguments=1)
+
+inDir <- sub("/*$","", opt$args[1])
+
+libMissing <- !require(parallel, quietly=T)
+if (libMissing) {stop("Failed to load R package 'parallel'")}
+
+
+### Function defs
+
+combineCounts <- function(inDir, treat, junc, cores=1) {
+### Reads individual raw well counts from the 'counts' folder and combine into one table
+
+    files <- list.files(file.path(inDir, "counts"), pattern=paste(treat$Batch[1], ".*W.*.counts.tab", sep=""), full.names=T)
+    fileBC <- sub(".*(W[0-9]+).*", "\\1", files)
+
+    if (length(files) == 0)                {stop("Raw well data not found")}
+    if (!(all(treat$Barcode %in% fileBC))) {stop("Not all raw well data files were found")}
+    if (!all(fileBC == treat$Barcode))     {stop("Mismatch betwetween treatment table and found raw well data files")}
+
+    ## Get total reads
+    mapFilesF <- dir(file.path(inDir, "map"), pattern=".*(R1|Fwd|fwd|Fw|fw)\\.stats\\.txt")
+    mapFilesR <- dir(file.path(inDir, "map"), pattern=".*(Rw|Rev|rev|Rv|rv)\\.stats\\.txt")    
+    statsF <- lapply(file.path(inDir, "map", mapFilesF), read.delim, header=F)
+    statsR <- lapply(file.path(inDir, "map", mapFilesR), read.delim, header=F)
+    statsF <- do.call("rbind", statsF)
+    statsR <- do.call("rbind", statsR)
+
+    bcF <- sub(".+(W[0-9]+).*", "\\1", statsF[,1])
+    bcR <- sub(".+(W[0-9]+).*", "\\1", statsR[,1])
+    if (!all(bcF == bcR))             {stop("Mismatch between fwd and rev read stats files")}
+    if (!all(treat$Barcode %in% bcF)) {stop("Not all expected mapping stats files were found")}    
+    names(statsF) <- names(statsR) <- c("file","unmapped","total","mappability")
+    rownames(statsF) <- rownames(statsR) <- sub("[^_]+_(.+)_[^_]+", "\\1", statsF[,1])
+    statsF <- statsF[match(treat$Barcode, bcF),]
+    statsR <- statsF[match(treat$Barcode, bcR),]
+
+    input  <- mclapply(files, read.csv, sep="\t", header=F, mc.cores=cores)
+    counts <- sapply(input, "[[", 4)
+    inputLabel <- paste(input[[1]][,1], input[[1]][,2], sep="_")
+    geneCounts <- aggregate(counts, by=list(Gene=paste(input[[1]][,1], substr(input[[1]][,2], 1, 2), sep=".")), FUN=sum)
+
+    if (any(!is.na(junc$Label))) {
+        collapseJunc <- junc[!is.na(junc$Label),]
+        juncF <- strsplit(collapseJunc$JunctionsFw, split=",")
+        juncR <- strsplit(collapseJunc$JunctionsRv, split=",")
+
+        collapseLabel <- rep(NA, nrow(input[[1]]))
+        for (i in 1:nrow(collapseJunc)) {
+            collapseLabel[input[[1]][,1] == collapseJunc$Gene[i] & input[[1]][,2] %in% juncF[[i]]] <-
+                paste0(collapseJunc$Gene[i], ".fw.", collapseJunc$Label[i])
+            collapseLabel[input[[1]][,1] == collapseJunc$Gene[i] & input[[1]][,2] %in% juncR[[i]]] <-
+                paste0(collapseJunc$Gene[i], ".rv.", collapseJunc$Label[i])
+        }
+        eventCounts <- aggregate(counts, by=list(Event=collapseLabel), FUN=sum)
+        eventCounts <- data.frame(Gene=sub("(.*)\\.[^.]+", "\\1", eventCounts$Event), eventCounts)
+        subtractCounts <- aggregate(eventCounts[,-c(1,2)], by=list(Gene=eventCounts$Gene), FUN=sum)
+
+        out <- geneCounts[!(geneCounts$Gene %in% subtractCounts$Gene),]
+        tmp <- geneCounts[match(subtractCounts$Gene, geneCounts$Gene),]
+        tmp <- data.frame(Gene=paste0(subtractCounts$Gene, ".RNA"),
+                          as.matrix(tmp[,-1]) - as.matrix(subtractCounts[,-1])
+                          )
+        out <- rbind(out,
+                     tmp,
+                     data.frame(Gene=sub("A1","GEN",eventCounts$Event), eventCounts[,-c(1,2)])
+                     )
+        out <- out[order(out$Gene),]
+    } else {
+        out <- geneCounts
+    }
+    outNames <- as.character(out$Gene)
+    out <- t(out[,-1])
+    colnames(out) <- outNames
+    rownames(out) <- treat$Barcode
+
+    out <- cbind(out,
+                 totReads.fw    = statsF[,"total"],
+                 totReads.rv    = statsR[,"total"],
+                 mappedReads.fw = statsF[,"total"] - statsF["unmapped"],
+                 mappedReads.rv = statsR[,"total"] - statsR["unmapped"],
+                 mappability.fw = round(100 * (1 - (statsF[,"unmapped"] / statsF[,"total"])), 2),
+                 mappability.rv = round(100 * (1 - (statsR[,"unmapped"] / statsR[,"total"])), 2)
+                 )
+
+    out
+}
+
+avgFRcounts <- function(x, junc, direction=c("both","fwd","rev")[1], imbal=1.2, imbalCts=10) {
+### Average forward and reverse read counts, or select one; Also check for imbalance more than 'imbal'
+    eventsFw <- sort(grep(".*\\.fw", colnames(x), value=T))
+    fwInd <- sapply(eventsFw, FUN=function(y) {grep(y, colnames(x))})
+    rvInd <- sapply(sub("\\.fw", ".rv", eventsFw), FUN=function(y) {grep(y, colnames(x))})
+
+    ## Check balance of fwd and rev
+    bal <- abs(log(10 + x[,fwInd]) / log(10 + x[,rvInd]))
+    meanFR <- (x[,fwInd] + x[,rvInd]) / 2
+    warn <- (bal < 1 - log(imbal) | bal > 1 + log(imbal)) & meanFR > imbalCts
+    if (any(as.vector(warn))) {
+        for (i in which(apply(warn, MAR=2, any))) {
+            warning(length(which(warn[,i])), " imbalanced samples for ", colnames(meanFR)[i])
+        }
+    }    
+
+    if (direction == "fwd") {
+        out <- x[,fwInd]
+        colnames(out) <- sub("\\.fw", "", colnames(out))
+    }
+    if (direction == "rev") {
+        out <- x[,rvInd]
+        colnames(out) <- sub("\\.rv", "", colnames(out))
+    }
+    if (direction == "both") {
+        if (is.list(fwInd) || is.list(rvInd)) {
+            stop("Forward and reverse read columns in raw counts file could not be matched for all events")
+        }
+
+        out <- round(meanFR)
+        colnames(out) <- sub("\\.fw", "", colnames(out))
+    }
+
+    out
+}
+
+main <- function(inDir, treat, junc, direction=c("both","fwd","rev")[1], cores=1, imbal=1.2, imbalCts=10) {
+### Collect mapped read numbers from individual samples and creat tables with
+### raw fwd and rev counts, average counts (if selected), and RPM
+
+    rawCounts <- combineCounts(inDir, treat, junc, cores=cores)
+    counts <- avgFRcounts(rawCounts, direction=direction, imbal=imbal,
+                          junc = junc)
+    tmp <- counts[,-which(colnames(counts) %in% c("mappability","totReads"))]
+    rpm <- round(1000000 * tmp / rowSums(tmp), 1)
+
+    treatCols <- unlist(sapply(c("ID","Barcode","Batch","Replicate","Treatment","Type","Status"),
+                               FUN=grep, x=names(treat)))
+    treat <- treat[,treatCols]
+
+    if (is.null(treat$Batch)) {
+        outName <- ""
+    } else {
+        outName <- paste0(treat$Batch[1], "_") 
+    }
+    dirTag <- paste0("_", direction)
+
+    outDir <- file.path(inDir, "expression")
+    if (!dir.exists(outDir)) {dir.create(outDir, recursive=TRUE)}
+
+    write.csv(data.frame(treat, rawCounts, check.names=F),
+              file=file.path(outDir, paste0(outName, "CountsRaw.csv")))
+    write.csv(data.frame(treat, counts, check.names=F),
+              file=file.path(outDir, paste0(outName, "Counts", dirTag, ".csv")), row.names=F)
+    write.csv(data.frame(treat, rpm, check.names=F),
+              file=file.path(outDir, paste0(outName, "RPM", dirTag, ".csv")), row.names=F)
+
+    cat("Read and RPM files saved in", outDir, "\n")
+}
+
+
+
+
+
+### Body
+
+## Check input
+if (length(opt$args) != 1)
+    stop(print_help(parser))
+if (is.null(opt$options$eventTab)) 
+    stop("eventTab must be provided")
+if (is.null(opt$options$treatTab)) 
+    stop("treatTab must be provided")
+if (!(opt$options$direction %in% c("both","fwd","rev"))) 
+    stop("direction must be one of 'both', 'fwd', or 'rev'")
+
+## Load tables
+treat <- read.delim(opt$options$treatTab)
+treat <- treat[order(treat$Barcode),]
+
+junc <- read.delim(opt$options$eventTab, as.is=T)
+
+
+## Body
+main(inDir, treat, junc,
+     direction = opt$options$direction,
+     cores     = opt$options$cores,
+     imbal     = opt$options$imbal,
+     imbalCts  = opt$options$imbal
+     )
+
+#opt <- list(
+#    options = list(
+#        treatTab  = "~/bin/SPARpipe/examples/COVID-19/SampleTable.tab",
+#        eventTab  = "~/bin/SPARpipe/examples/COVID-19/amplicons/EventJunctions.tab",
+#        direction = "fwd",
+#        imbal     = 1.5,
+#        imbalCts  = 20,
+#        cores     = 1
+#    ),
+#    args    = "~/bin/SPARpipe/examples/COVID-19/test_align/"
+#)
+

examples/COVID-19/PoC_ReadCount_Table.csv

@@ -0,0 +1,27 @@
+fastq-file/sample name,PPIB,Ngene,Egene,RdRP,Sgene.RBD,Sgene.Poly,mapping%
+W609_HEK293T-rep1,49152,0,1,1,3,2,90.85
+W621_HEK293T-rep2,55368,5,0,2,2,0,96.94
+W633_LTRI-20,23254,3,0,2,1,0,74.53
+W645_LTRI-13,7400,1,0,0,1,1,77.05
+W657_LTRI-35,11530,24023,3082,9911,15318,19653,98.47
+W669_LTRI-36,11876,16764,1670,5815,8680,12000,98.36
+W586_LTRI-32,49078,1752,146,222,1844,2198,97.57
+W598_LTRI-28,5296,18336,3057,5551,6508,9692,90.48
+W610_LTRI-27,0,8258,156,1392,4666,6028,96.22
+W622_LTRI-33,24556,7527,502,1230,3654,1032,97.71
+W634_LTRI-21,38302,7424,246,900,2002,4078,96.76
+W646_LTRI-22,906,34718,3520,9420,14792,22242,97.48
+W658_LTRI-34,1621,27632,3038,7954,13114,18448,98.09
+W670_LTRI-24,2158,19434,2070,5063,9800,13128,98.35
+W587_LTRI-26,279,31589,3312,8568,15040,22452,98.61
+W599_LTRI-29,134,31968,4871,11048,15924,25968,98.52
+W611_LTRI-30,507,27292,3476,9857,14336,20062,98.45
+W623_LTRI-23,264,19518,3022,5348,11004,15160,98.48
+W635_LTRI-31,20,17220,4682,8928,14429,19388,98.49
+W647_LTRI-25,24,16955,5802,12460,15156,20241,98.52
+W659_LTRI-18,3186,12088,3084,6093,8923,11260,98.25
+W671_Twist2:-dil1,3,15862,4494,9988,14120,18706,97.43
+W588_Twist2-dil2,1,14780,2266,6610,9346,14248,97.2
+W600_Twist2-dil3,2,11194,4024,7262,8932,10612,96.77
+W612_Twist2-dil4,1,9354,830,2240,5562,10107,93.83
+W624_Twist2-dil5,0,0,202,0,797,4300,49.6

examples/COVID-19/SampleTable.tab

@@ -0,0 +1,27 @@
+ID	Replicate	Batch	Barcode	Treatment
+S163	R1	200423	W609	HEK293T-Qiagen_MIXE_30x
+S164	R1	200423	W621	HEK293T-Norgen_MIXE_30x
+S165	R1	200423	W633	LTRI-20_MIXE_30x
+S166	R1	200423	W645	LTRI-13_MIXE_30x
+S167	R1	200423	W657	LTRI-35_MIXE_30x
+S168	R1	200423	W669	LTRI-36_MIXE_30x
+S169	R1	200423	W586	LTRI-32_MIXE_30x
+S170	R1	200423	W598	LTRI-28_MIXE_30x
+S171	R1	200423	W610	LTRI-27_MIXE_30x
+S172	R1	200423	W622	LTRI-33_MIXE_30x
+S173	R1	200423	W634	LTRI-21_MIXE_30x
+S174	R1	200423	W646	LTRI-22_MIXE_30x
+S175	R1	200423	W658	LTRI-34_MIXE_30x
+S176	R1	200423	W670	LTRI-24_MIXE_30x
+S177	R1	200423	W587	LTRI-26_MIXE_30x
+S178	R1	200423	W599	LTRI-29_MIXE_30x
+S179	R1	200423	W611	LTRI-30_MIXE_30x
+S180	R1	200423	W623	LTRI-23_MIXE_30x
+S181	R1	200423	W635	LTRI-31_MIXE_30x
+S182	R1	200423	W647	LTRI-25_MIXE_30x
+S183	R1	200423	W659	LTRI-18_MIXE_30x
+S184	R1	200423	W671	Twist2:50000_MIXE_30x
+S185	R1	200423	W588	Twist2:5000_MIXE_30x
+S186	R1	200423	W600	Twist2:500_MIXE_30x
+S187	R1	200423	W612	Twist2:50_MIXE_30x
+S188	R1	200423	W624	Twist2:5_MIXE_30x

examples/COVID-19/ToDo

@@ -0,0 +1,13 @@
+../../accessories/MakeJunctionsFASTA.R -e amplicons/JunctionLibraryElements_test200423.csv -p primers/PrimerChecking_SPARseq.optimized.COVD19_200423.csv -l 75
+
+mkdir junctions/bowtie_indices
+bowtie-build junctions/Junctions_fwd.fa junctions/bowtie_indices/Junctions_fwd
+bowtie-build junctions/Junctions_rev.fa junctions/bowtie_indices/Junctions_rev
+
+
+for f in fastq_files/200423_W*Fwd.fq.gz; do ../../2_align.pl --junc=amplicons/bowtie_indices/Junctions_fwd --outDir=test_align --trim3=1 --btopt="--best -n 3 -k 1 -m 1" ${f}; done
+for f in fastq_files/200423_W*Rev.fq.gz; do ../../2_align.pl --junc=amplicons/bowtie_indices/Junctions_rev --outDir=test_align --trim3=1 --btopt="--best -n 3 -k 1 -m 1" ${f}; done
+
+../../3_combine.pl --juncBase=amplicons/Junctions --eventTab=amplicons/EventJunctions.tab --treatTab=SampleTable.tab test_align
+
+### Combine into one read table

examples/COVID-19/amplicons/EventJunctions.tab

@@ -0,0 +1,8 @@
+Gene	Event	Label	Relative	JunctionsFw	JunctionsRv
+004.PPIB	NA	NA	NA	fw1	rv1
+006.Ngene	NA	NA	NA	fw1	rv1
+008.Egene	NA	NA	NA	fw1	rv1
+010.RdRP	NA	NA	NA	fw1	rv1
+013.Sgene.RBD	NA	NA	NA	fw1	rv1
+014.Sgene.Poly	NA	NA	NA	fw1	rv1
+019.ACTB	NA	NA	NA	fw1	rv1

examples/COVID-19/amplicons/JunctionLibraryElements_test200423.csv

@@ -0,0 +1,8 @@
+"gene","origin","event","structure","chrom","strand","C1.start","C1.end","C2.start","C2.end","C1.seq","C2.seq"
+"004.PPIB","background","004.PPIP.ctrlM","C1-C2","chr15","-",64162852,64163155,64162041,64162154,"ACTATCCGGCGCCGAGCCGGAGGGGGGAAACGGcgcccgccgcccgcccggagcccgcgagcaaccccagtcccccccacccgcgcgTGGCGGCGCCGGCTCCCTAGCCACCGCGGCCCCACCCTCTTCCGGCCTCAGCTGTCCGGGCTGCTTTCGCCTCCGCCTGTGGATGCTGCGCCTCTCCGAACGCAACATGAAGGTGCTCCTTGCCGCCGCCCTCATCGCGGGGTCCGTCTTCTTCCTGCTGCTGCCGGGACCTTCTGCGGCCGATGAGAAGAAGAAGGGGCCCAAAGTCACCGTCAAG","GTGTATTTTGACCTACGAATTGGAGATGAAGATGTAGGCCGGGTGATCTTTGGTCTCTTCGGAAAGACTGTTCCAAAAACAGTGGATAATTTTGTGGCCTTAGCTACAGGAGAG"
+"006.Ngene","foreground","006.Ngene.SPARseq1","C1-C2","NC_045512.2","+",28867,28938,28939,29010,"TCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCT","GCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGT"
+"008.Egene","foreground","008.Egene.SPARseq1","C1-C2","NC_045512.2","+",26203,26346,26347,26418,"ACTAGCGTGCCTTTGTAAGCACAAGCTGATGAGTACGAACTTATGTACTCATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTT","ACTGCGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTCTTGTAAAACCTTCTTTTTACGTT"
+"010.RdRP","foreground","010.RdRP.SPARseq1","C1-C2","NC_045512.2","+",15406,15477,15478,15549,"GCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGA","ACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAAT"
+"013.Sgene.RBD","foreground","013.Sgene.RBD.SPARseq1","C1-C2","NC_045512.2","+",22963,23034,23035,23106,"TATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCC","TTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTT"
+"014.Sgene.Poly","foreground","014.Sgene.Poly.SPARseq1","C1-C2","NC_045512.2","+",23539,23610,23611,23754,"ATATGAGTGTGACATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCG","GGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGCCATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGTCTAT"
+"019.ACTB","background","019.ACTB.ctrl.SPARseq2","C1-C2","chr7","-",5528281,5528719,5528004,5528185,"ATCATGTTTGAGACCTTCAACACCCCAGCCATGTACGTTGCTATCCAGGCTGTGCTATCCCTGTACGCCTCTGGCCGTACCACTGGCATCGTGATGGACTCCGGTGACGGGGTCACCCACACTGTGCCCATCTACGAGGGGTATGCCCTCCCCCATGCCATCCTGCGTCTGGACCTGGCTGGCCGGGACCTGACTGACTACCTCATGAAGATCCTCACCGAGCGCGGCTACAGCTTCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGCTACGTCGCCCTGGACTTCGAGCAAGAGATGGCCACGGCTGCTTCCAGCTCCTCCCTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATGAGCGGTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCCTGG","GCATGGAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGTGACGTGGACATCCGCAAAGACCTGTACGCCAACACAGTGCTGTCTGGCGGCACCACCATGTACCCTGGCATTGCCGACAGGATGCAGAAGGAGATCACTGCCCTGGCACCCAGCACAATGAAGATCAAG"

examples/COVID-19/amplicons/Junctions_amplicons.fa

@@ -0,0 +1,14 @@
+>004.PPIB_C1C2
+TGCTGCTGCCGGGACCTTCTGCGGCCGATGAGAAGAAGAAGGGGCCCAAAGTCACCGTCAAGGTGTATTTTGACCTACGAATTGGAGATGAAGATGTAGGCCGGGTGATCTTTGGTCTCTTCGGAA
+>006.Ngene_C1C2
+CCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAA
+>008.Egene_C1C2
+AGACAGGTACGTTAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTTACTGCGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTG
+>010.RdRP_C1C2
+TGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCAC
+>013.Sgene.RBD_C1C2
+ATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGT
+>014.Sgene.Poly_C1C2
+TATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTAC
+>019.ACTB_C1C2
+TCACCATTGGCAATGAGCGGTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCCTGTGGCATCCACGAAACTACCTTCAACTCCATCATGAAGTGTGACGTGG

examples/COVID-19/amplicons/Junctions_fwd.bed

@@ -0,0 +1,7 @@
+004.PPIB_fw1_C1C2	0	75
+006.Ngene_fw1_C1C2	0	75
+008.Egene_fw1_C1	0	75
+010.RdRP_fw1_C1C2	0	75
+013.Sgene.RBD_fw1_C1C2	0	75
+014.Sgene.Poly_fw1_C1C2	0	75
+019.ACTB_fw1_C1C2	0	75

examples/COVID-19/amplicons/Junctions_fwd.fa

@@ -0,0 +1,14 @@
+>004.PPIB_fw1_C1C2
+TGCTGCTGCCGGGACCTTCTGCGGCCGATGAGAAGAAGAAGGGGCCCAAAGTCACCGTCAAGGTGTATTTTGACC
+>006.Ngene_fw1_C1C2
+CCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTG
+>008.Egene_fw1_C1
+AGACAGGTACGTTAATAGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCA
+>010.RdRP_fw1_C1C2
+TGAGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTG
+>013.Sgene.RBD_fw1_C1C2
+ATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCC
+>014.Sgene.Poly_fw1_C1C2
+TATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCT
+>019.ACTB_fw1_C1C2
+TCACCATTGGCAATGAGCGGTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCCTGGGCATGGAGTCCTGTG

examples/COVID-19/amplicons/Junctions_fwd_minDist.tab

@@ -0,0 +1,8 @@
+junction	distEvent	distAll
+004.PPIB_fw1_C1C2	NA	54
+006.Ngene_fw1_C1C2	NA	53
+008.Egene_fw1_C1	NA	49
+010.RdRP_fw1_C1C2	NA	49
+013.Sgene.RBD_fw1_C1C2	NA	52
+014.Sgene.Poly_fw1_C1C2	NA	52
+019.ACTB_fw1_C1C2	NA	53

examples/COVID-19/amplicons/Junctions_rev.bed

@@ -0,0 +1,7 @@
+004.PPIB_rv1_C2C1	0	75
+006.Ngene_rv1_C2C1	0	75
+008.Egene_rv1_C2C1	0	75
+010.RdRP_rv1_C2C1	0	75
+013.Sgene.RBD_rv1_C2C1	0	75
+014.Sgene.Poly_rv1_C2C1	0	75
+019.ACTB_rv1_C2C1	0	75