One-to-N sequence matches in coronavirus genomes

#### General parameters for the analysis ####
reloadImage <- FALSE
quickTest <- FALSE # if True, only analyse the 2 first features


#### Sequence collection ####

## Supported: 
collections <- c(
  "around-CoV-2", 
  "selected",
  "around-CoV-2-plus-GISAID", 
  "selected-plus-GISAID"
  )



# collection <- "around-CoV-2" # ~20 strains
# collection <- "all" # ~60 strains
# collection <- "selected" # ~50 strains
# collection <- "around-CoV-2-plus-GISAID" # 16 strains
collection <- "selected-plus-GISAID" # ~40 strains
# collection <- "all-plus-GISAID" # ~60 strains



## Choose a collection-specific path for the figures
knitr::opts_chunk$set(fig.path = paste0('figures/one-to-n-matches_', collection, '/', collection, "_"))

## Note about GIDAID sequences.
## 
## A few genomes were not available in NCBI Genbank at the time of 
## this analysis, and had to be downloaded from GISAID. These sequences
##  can however not be redistributed, they should thus be downloaded 
##  manually to reproduce the full trees. 


## Exclude incomplete genomes (i.e. those containing a lof of Ns) to avoid biases in the distance computations
excludeIncomplete <- TRUE



#### Define directories and files ####
dir <- list(main = '..')
dir$R <- file.path(dir$main, "scripts/R")

## Input directory for sequences
dir$seqdata <- file.path(dir$main, "data")
dir.create(dir$seqdata, showWarnings = FALSE, recursive = TRUE)


## Create output directory for aligned sequences
dir$results <- file.path(dir$main, "results")
dir.create(dir$results, showWarnings = FALSE, recursive = TRUE)

## Instantiate a list for output files
outfiles <- vector()

## Memory image
dir$images <- file.path(dir$main, "memory_images")
dir.create(dir$images, recursive = TRUE, showWarnings = FALSE)
outfiles["Memory image"] <- file.path(
  dir$images, 
  paste0(collection, "_one-to-n_matches.Rdata"))

## Input files
infiles <- list()

## Output tables
# di$output <- file.path(dir.main, "")
# dir$tables <- 

## Load custom functions
source(file.path(dir$R, "align_n_to_one.R"))
source(file.path(dir$R, "plot_pip_profiles.R"))
source(file.path(dir$R, "plot_pip_profiles_from_list.R"))

## A unequivocal pattern to identify the reference genome in the sequence names of the input file
refPattern <- "HuCoV2_WH01_2019"

## Exclude some genomes with a lot of Ns, because they bias the PIP profiles and alignments and trees
excludePatterns <- c("PnGu-P2S_2019")




#### Features of interest in the reference genome ####

features <- list()


#### Specific features  ####

## Cited from Sun et al (https://doi.org/10.1016/j.molmed.2020.02.008)in Lin et al. https://doi.org/10.20944/preprints202006.0044.v1

features[['Sun_2020']]  <- c(start = 13522, end =  23686, isCDS = FALSE)

## S1, first cleavage product of the spike protein
features[['S1']] <- c(start = 21599, end = 23617, isCDS = TRUE)

## Receptor binding domain
features[['RBD']] <- c(start = 22517, end = 23185, isCDS = TRUE)

## S2, second cleavage product of the spike protein
features[['S2']] <- c(start = 23618, end = 25381, isCDS = TRUE)



## Regions around the insertions
features[['Ins1-pm120']] <- c(start = 21647, end = 21907, isCDS = FALSE)
features[['Ins2-pm120']] <- c(start = 21899, end = 22156, isCDS = FALSE)
features[['Ins3-pm120']] <- c(start = 21899, end = 23152, isCDS = FALSE)
features[['Ins4-pm120']] <- c(start = 23483, end = 23734, isCDS = FALSE)
features[['Ins4-m240']] <- c(start = 23363, end = 23614, isCDS = FALSE)

## Receptor-binding domain

## Potential Pangolin origin after Xiao (https://doi.org/10.1101/2020.02.17.951335)
features[['Recomb-Xiao']] <- c(start = 22871, end = 23092, isCDS = FALSE)

## Recombinant region 1 seen on the PIP profiles
features[['Recomb-reg-1']] <- c(start = 2900, end = 3800, isCDS = FALSE)

## Recombinant region 2 seen on the PIP profiles
features[['Recomb-reg-2']] <- c(start = 21000, end = 24000, isCDS = FALSE)


## Recombinant region 3 seen on the PIP profiles
features[['Recomb-reg-3']] <- c(start = 27500, end = 28550, isCDS = FALSE)

## Recombinant region 4 in the RBD
## Slighty wider than Xiao's limits in order to see the context
features[['Recomb-RBD']] <- c(start = 22700, end = 23200, isCDS = FALSE)
# features[['Recomb-RBD']] <- c(start = 22760, end = 23160, isCDS = FALSE)

## Annotated coding sequences
features[['CDS-S']] <- c(start = 21563, end = 25384, isCDS = TRUE) ## Spike gene
features[['CDS-ORF3a']] <- c(start = 25393, end = 26220, isCDS = TRUE)
features[['CDS-E']] <- c(start = 26245, end = 26472, isCDS = TRUE)
features[['CDS-M']] <- c(start = 26523, end = 27191, isCDS = TRUE)
features[['CDS-ORF6']] <- c(start = 27202, end = 27387, isCDS = TRUE)
features[['CDS-ORF7a']] <- c(start = 27394, end = 27759, isCDS = TRUE)
features[['CDS-ORF8']] <- c(start = 27894, end = 28259, isCDS = TRUE)
features[['CDS-N']] <- c(start = 28274, end = 29533, isCDS = TRUE)
features[['CDS-ORF10']] <- c(start = 29558, end = 29674, isCDS = TRUE)

## Large ORF covering 2 thirds of the genome, coding for polyprotein
features[['CDS-ORF1ab']] <- c(start = 266, end = 21555, isCDS = TRUE)

## All the sequences after the big polyprotein-coding ORF1ab
features[['After-ORF1ab']] <- c(start = 21556, end = 29899, isCDS = FALSE)

## Only keep the two first features if quick test has been specified
if (quickTest) {
  features <- features[1:2]
}

## Convert feature list into a feature tbale
featureTable <- as.data.frame(t(as.data.frame.list(
  features, stringsAsFactors = FALSE)),
  row.names = names(features)  # to avoid replacement of "-" by "."
  )

# class(featureTable)
featureTable$length <- featureTable$end - featureTable$start + 1
# row.names(featureTable)
# featureTable["CDS.S", ]

featureTable <- featureTable[order(featureTable$start), ]


message("Analysing ", length(features), " features")
message("\t", paste(collapse = ", ", names(features)))

## Report the parameters
message("\tReference pattern: ", refPattern)

Strain collection: selected-plus-GISAID

## Genome dir and files
if (length(grep(pattern = "GISAID", x = collection)) > 0) {
  useGISAID <- TRUE
  dir$sequences <- file.path(dir$main, "data", "GISAID_genomes")
  # collections <-  paste0(collections, "-plus-GISAID")
  # collection <-  paste0(collection, "-plus-GISAID")
} else {
  dir$sequences <- file.path(dir$main, "data", "genomes")
}


## Define the input sequences
infiles$sequences <- file.path(
  dir$sequences, 
  paste0("genomes_", collection, ".fasta"))

## Check if the input  sequence file exists
if (!file.exists(infiles$sequences)) {
  stop("Genome sequence file is missing", "\n", infiles$sequences)
} else {
  message("Genome sequence file", "\n", infiles$sequences)
}

#### Load genome sequences ####
genomes <- readDNAStringSet(filepath = infiles$sequences, format = "fasta")

## Shorten sequence names by suppressing the fasta comment (after the space)
names(genomes) <- sub(pattern = " .*", replacement = "", x = names(genomes), perl = TRUE)

## Exclude genomes
if (excludeIncomplete) {
  excludePattern = paste0("(", paste(collapse = ")|(", excludePatterns), ")")
  excludedstrainNames <- grep(pattern = excludePattern, x = names(genomes), 
                              value = TRUE, invert = FALSE)
  filteredGenomeIndices <- grep(pattern = excludePattern, x = names(genomes), 
                                value = FALSE, invert = TRUE)
  message("\tExcluded ", length(excludedstrainNames)," genomes: ", paste(collapse = ", ", excludedstrainNames)) 
  message("\tRemaining genomes: ", length(filteredGenomeIndices))
  genomes <- genomes[filteredGenomeIndices]
# names(genomes)
}

## Report the number of genoomes
strainNames <- names(genomes)
nbStrains <- length(strainNames)
message("\tLoaded ", nbStrains, " genomes from file ", infiles$sequences)
# View(genomes)


#### Define reference and query genomes ####
refStrainName <- grep(pattern = refPattern, x = names(genomes), 
                      ignore.case = TRUE, value = TRUE)
if (is.null(refStrainName)) {
  stop("Could not identify reference genome with pattern ", refPattern)
}
message("\tReference genome name: ", refStrainName)

allFeatureAlignments <- list()

## Reload memory image if specified in parameters
if (reloadImage) {
  load(outfiles["Memory image"])
  reloadImage <- TRUE
}

Parameters

#### Define a list of parameters ####
parameters <- list()
parameters$collection <- collection
parameters$refStrainName <- refStrainName
parameters$nbStrains <- nbStrains
parameters$sequenceDir <- dir$sequences
parameters$sequenceFile <- infiles$sequences
parameters$memoryImage <- outfiles["Memory image"]


## Exclude metagenomic assemblies
parameters$pasDeTambouille <- FALSE
if (parameters$pasDeTambouille) {
  excludePatterns <- append(excludePatterns, "BtYu-RmYN02_2019")
}

kable(t(as.data.frame.list(parameters)), 
      caption = "Parameters of the analysis",
      col.names = "Parameter")

Parameters of the analysis
	Parameter
collection	selected-plus-GISAID
refStrainName	HuCoV2_WH01_2019
nbStrains	47
sequenceDir	../data/GISAID_genomes
sequenceFile	../data/GISAID_genomes/genomes_selected-plus-GISAID.fasta
memoryImage	../memory_images/selected-plus-GISAID_one-to-n_matches.Rdata
pasDeTambouille	FALSE

Strain statistics

#### Compute statistics about sequence sizes ###
strainStats <- data.frame(
  n = 1:length(genomes),
  row.names = names(genomes),
  status = rep("Query", length.out = length(strainNames))
)
strainStats[,"status"] <- as.vector(strainStats[,"status"])
strainStats[refStrainName,"status"] <- "Reference"
g <- 1
for (g in strainNames) {
  strainStats[g, "length"] <- length(genomes[[g]])
}

#### Define the color associated to each sequence ####


## Color palette per species
speciesPalette <- list(
  Human = "#880000",
  Bat = "#888888",
  Pangolin = "#447700",
  Camel = "#BB8800",
  Pig = "#FFBBBB",
  Civet = "#00BBFF"
)

## Species prefix in the tip labels
speciesPrefix <- c("Hu" = "Human",
                   "Bt" = "Bat",
                   "Pn" = "Pangolin",
                   "Cm" = "Camel",
                   "Pi" = "Pig",
                   "Cv" = "Civet")

## Strain-specific colors
strainColor <- c(
  "HuCoV2_WH01_2019" = "red",
  "HuSARS-Frankfurt-1_2003" = "#0044BB",
  "PnGu1_2019" = "#00CC00",
  "PnMP789" = "#00FF88",
  "BtRaTG13_" = "#FF6600",
  "BtYu-RmYN" = "#FFBB22",
  "BtZXC21" = "black", 
  "BtZC45" = "black")

## Identify species per tip
for (prefix in names(speciesPrefix)) {
  strainStats[grep(pattern = paste0("^", prefix), x = row.names(strainStats), perl = TRUE), "species"] <- speciesPrefix[prefix]
  
}

## Assign acolor to each species
strainStats$color <- "grey" # default
strainStats$color <- speciesPalette[as.vector(strainStats$species)]

for (strain in names(strainColor)) {
  strainStats[grep(pattern = paste0("^", strain), 
                  x = row.names(strainStats), perl = TRUE), "color"] <- strainColor[strain]
  
}


## Assign specific color to some nodes

## Define a color for each strain
strainColors <- (unlist(strainStats$color))
names(strainColors) <- row.names(strainStats)

kable(strainStats, caption = "Reference and query genomes")

Reference and query genomes
	n	status	length	species	color
BtBM48-31	1	Query	29276	Bat	#888888
BtBtKY72	2	Query	29274	Bat	#888888
BtCp-Yun_2011	3	Query	29452	Bat	#888888
BtGX2013	4	Query	29161	Bat	#888888
BtHKU3-12	5	Query	29704	Bat	#888888
BtHKU5	6	Query	30482	Bat	#888888
BtHKU9-1	7	Query	29114	Bat	#888888
BtJL2012	8	Query	29037	Bat	#888888
BtLYRa11	9	Query	29805	Bat	#888888
BtRaTG13_2013_Yunnan	10	Query	29855	Bat	#FF6600
Btrec-SARSg_2008	11	Query	29750	Bat	#888888
BtRm1/2004	12	Query	29749	Bat	#888888
BtRp-Shaanxi2011	13	Query	29484	Bat	#888888
BtRp3-2004	14	Query	29736	Bat	#888888
BtRs_672-2006	15	Query	29059	Bat	#888888
BtRs4874	16	Query	30311	Bat	#888888
BtSC2018	17	Query	29648	Bat	#888888
BtYN2013	18	Query	29142	Bat	#888888
BtYN2018B	19	Query	30256	Bat	#888888
BtYN2018C	20	Query	29689	Bat	#888888
BtYNLF_31C	21	Query	29723	Bat	#888888
BtZC45	22	Query	29802	Bat	black
BtZXC21	23	Query	29732	Bat	black
CmMERS	24	Query	29851	Camel	#BB8800
Cv007-2004	25	Query	29540	Civet	#00BBFF
Hu229E	26	Query	27317	Human	#880000
HuCoV2_WH01_2019	27	Reference	29899	Human	red
HuMERS_172-06_2015	28	Query	30068	Human	#880000
HuNL63	29	Query	27553	Human	#880000
HuOC43	30	Query	30741	Human	#880000
HuSARS-Frankfurt-1_2003	31	Query	29727	Human	#0044BB
HuTGEV	32	Query	28586	Human	#880000
PiPRCV	33	Query	27765	Pig	#FFBBBB
PiSADS	34	Query	27163	Pig	#FFBBBB
PnGX-P1E_2017	35	Query	29801	Pangolin	#447700
PnGX-P2V_2018	36	Query	29795	Pangolin	#447700
PnMP789	37	Query	29521	Pangolin	#00FF88
BtRc-o319	38	Query	29718	Bat	#888888
BtRacCS203	39	Query	29832	Bat	#888888
BtRacCS264	40	Query	29820	Bat	#888888
BtRacCS253	41	Query	29820	Bat	#888888
BtRacCS224	42	Query	29820	Bat	#888888
BtRacCS271	43	Query	29820	Bat	#888888
BtYu-RmYN02_2019	44	Query	29671	Bat	#FFBB22
PnGu1_2019	45	Query	29825	Pangolin	#00CC00
BtCambodia/RShSTT200/2010	46	Query	29793	Bat	#888888
BtCambodia/RShSTT182/2010	47	Query	29787	Bat	#888888

The collection selected-plus-GISAID contains 47 virus genome sequences.

Features

## Print feature coordinates
kable(featureTable, caption = "Feature coordinates. ")

Feature coordinates.
	start	end	isCDS	length
CDS-ORF1ab	266	21555	1	21290
Recomb-reg-1	2900	3800	0	901
Sun_2020	13522	23686	0	10165
Recomb-reg-2	21000	24000	0	3001
After-ORF1ab	21556	29899	0	8344
CDS-S	21563	25384	1	3822
S1	21599	23617	1	2019
Ins1-pm120	21647	21907	0	261
Ins2-pm120	21899	22156	0	258
Ins3-pm120	21899	23152	0	1254
RBD	22517	23185	1	669
Recomb-RBD	22700	23200	0	501
Recomb-Xiao	22871	23092	0	222
Ins4-m240	23363	23614	0	252
Ins4-pm120	23483	23734	0	252
S2	23618	25381	1	1764
CDS-ORF3a	25393	26220	1	828
CDS-E	26245	26472	1	228
CDS-M	26523	27191	1	669
CDS-ORF6	27202	27387	1	186
CDS-ORF7a	27394	27759	1	366
Recomb-reg-3	27500	28550	0	1051
CDS-ORF8	27894	28259	1	366
CDS-N	28274	29533	1	1260
CDS-ORF10	29558	29674	1	117

Feature matches and alignments

We perform a global pairwise alignment (Needle-Waterman algorithm) between each feature of the reference strain (HuCoV2_WH01_2019) and each one of the query genomes.
The collected nucleic sequences are then aligned based on their translated sequences. This ensures a more relevant placing of the gaps. These translation-based alignments are exported in aminoacids and DNA formats (the DNA sequences are obtained by reverse-mapping the aligned aminoacid sequences onto the input DNA sequences).
For each alignment (AA and DNA) wee compute a consensus matrix, indicating the frequency of each residue (row of the consensus matrix) in each position of the alignment (column of the matrix).

#### One-to-N alignmemnt of user-speficied genomic features ####


i <- 1
testFeature <- "Recomb-RBD"
i <- which(names(features) == testFeature)

## Rune one-ton matches and translation-based mumti^ple alignment + PIP profiles
for (i in 1:length(features)) {
  featureName <- names(features)[i]
  
  ## Compute feature length
  featureLimits <- features[[featureName]]
  featureStart <- featureLimits[["start"]]
  featureEnd <- featureLimits[["end"]]
  featureLength <- 
    featureEnd - featureStart  + 1
  
  message("Searching matches for feature ", 
          i, "/", length(features), "\t",
          featureName, 
          " (", featureLimits[1], "-", featureLimits[2], ")", 
          " in collection ", collection)
  
  ## Print a section title with the feature name and limits
  cat(sep = "", "\n### ",  featureName, 
      " (", featureLimits[1], "-", featureLimits[2], 
      "; ", featureLength,"bp)", "\n")


  ## Choose a collection- and feature-specific path for the figures
  knitr::opts_chunk$set(fig.path = paste0(
    'figures/one-to-n-matches_', collection, '/',
    collection, '_vs_', featureName))

  
  #### Define feature-specific output directory ####
  featurePrefix <- paste0(
    featureName,
    "_", collection)
  
  dir[[featureName]] <- file.path(dir$results, featurePrefix)
  message("\tOutput directory: ", dir[[featureName]] )
  dir.create(dir[[featureName]], showWarnings = FALSE, recursive = TRUE)
  
  #### Find feature matches by 1-to-1 alignment with each genome ####

  ## Define output file for unaligned matching sequences
  outfiles[paste0(featurePrefix, "_matches")]  <- file.path(
    dir[[featureName]], paste0(featurePrefix, "_matches.fasta"))
  message("\tMatching sequences: ", 
          outfiles[paste0(featurePrefix, "_matches")])
  
  ## Get sequence of the feature from the reference genome
  refSeq <- subseq(genomes[refStrainName], 
                   start = featureLimits[1], 
                   end = featureLimits[2])

  ## Match the reference feature with all the genomes
  if (reloadImage) {
    featureAlignmentsNto1 <- allFeatureAlignments[[featureName]]
  } else {  
    featureAlignmentsNto1 <- alignNtoOne(
      refSequence = refSeq, 
      querySequences = genomes, 
      type = "global-local",
      outfile = outfiles[paste0(featurePrefix, "_matches")])
    allFeatureAlignments[[featureName]] <- featureAlignmentsNto1
    
  }
  
  
  
   
  
  
  #### Translation-based alignments ####
  if (featureTable[featureName, "isCDS"]) {
    message("\tTranslation-based multiple alignment of feature ", featureName, "; DNA output")
    
    #### Align translated sequenecs and return correponding nucleic sequences ####
    featureAlignmentsNto1$tralignedDNA <- AlignTranslation(
      DNAStringSet(featureAlignmentsNto1$sequences), 
      type = "DNAStringSet",
      verbose = FALSE)
    
    ## Save multiple-aligned DNA sequences
    outfiles[paste0(featurePrefix, "_tralignedDNA")]  <- file.path(
      dir[[featureName]], paste0(featurePrefix, "_tralignedDNA.fasta"))
    message("\tTranslation-based alignment saved in file ", 
            outfiles[paste0(featurePrefix, "_tralignedDNA")])
    writeXStringSet(
      x = featureAlignmentsNto1$tralignedDNA, 
      filepath = outfiles[paste0(featurePrefix, "_tralignedDNA")] )

    ## Convert alignment to position-weight matrix
    featureAlignmentsNto1$tralignedDNAConsensus <- consensusMatrix(
      x = featureAlignmentsNto1$tralignedDNA)
    
    #### Align translated sequenecs and return correponding AA sequences ####
    message("\tTranslation-based multiple alignment of feature ", featureName, "; AA output")
    featureAlignmentsNto1$tralignedAA <- AlignTranslation(
      DNAStringSet(featureAlignmentsNto1$sequences), 
      type = "AAStringSet",
      verbose = FALSE)

    ## Convert alignment to position-weight matrix
    featureAlignmentsNto1$tralignedAAConsensus <- consensusMatrix(
      x = featureAlignmentsNto1$tralignedAA)
  
    ## Save multiple-aligned AA sequences
    outfiles[paste0(featurePrefix, "_tralignedAA")]  <- file.path(
      dir[[featureName]], paste0(featurePrefix, "_tralignedAA.fasta"))
    message("\tTranslation-based alignment saved in file ", 
            outfiles[paste0(featurePrefix, "_tralignedAA")])
    writeXStringSet(
      x = featureAlignmentsNto1$tralignedAA, 
      filepath = outfiles[paste0(featurePrefix, "_tralignedAA")] )
  }
  
  ## Compute sequence order by decreasing PIP score
  seqOrder <- order(featureAlignmentsNto1$stat$score, decreasing = TRUE)
  

  ## Print matching statistics
  kable(featureAlignmentsNto1$stats[seqOrder, ], 
        caption = paste0(
          "One-to-N alignment of feature ", 
          featureName))
  
  #### PIP profiles ####
  ## Choose window size  
  if (featureLength > 20000) {
    windowSize <- 800 
  } else if (featureLength > 5000) {
    windowSize <- 500 
  } else if (featureLength > 3000) {
    windowSize <- 200 
  } else if (featureLength > 400) {
    windowSize <- 100 
  } else {
    windowSize <- max(50, 10 * round(featureLength / 100))
  }

  
  ## DNA PIP profile of one-to-N alignment
  PlotPIPprofilesFromList(
#    alignments = featureAlignmentsNto1$alignments[seqOrder],
    alignments = featureAlignmentsNto1$alignments,
    reversePlot = TRUE,
    windowSize = windowSize,
    main = paste0("PIP profiles: collection ", collection,
                  "\nGenomic feature ", featureName, " (", featureStart, ":", featureEnd ,", ", featureLength, " bp)"),
    #    colors = NULL,
    colors = strainColors,
    legendMargin = 0.25,
    legendCorner = "topright", 
    lwd = 1,
    legendCex = 0.5, 
    ylim = c(30, 100))

  ## Identify the correspodences between matches and sequences
  seqNames <- names(featureAlignmentsNto1$alignments)
  refSeqName <- refStrainName
  matchNames <- names(featureAlignmentsNto1$sequences) 
  refMatchName <- grep(pattern = paste0("^", refStrainName), matchNames, 
                       perl = TRUE, value = TRUE)

  ## Define colors
  matchColors <- strainColors[seqNames]
  names(matchColors) <- matchNames
  
  if (featureTable[featureName, "isCDS"]) {
    
    #### DNA PIP profile of translation-based multiple alignment ####
    PlotPIPprofiles(
      alignment = featureAlignmentsNto1$tralignedDNA, 
      refSeqName = refMatchName,
      plotRefGaps = FALSE,
      reversePlot = TRUE,
      windowSize = windowSize,
      main = paste0("DNA PIP profiles – collection: ", collection,
                    "\nGenomic feature ", featureName, " (", featureStart, ":", featureEnd ,", ", featureLength, " bp)"),
      colors = matchColors,
      legendMargin = 0.3,
      legendCorner = "topright",
      lwd = 1,
      legendCex = 0.5,
      ylim = c(30, 100))
    
    ## AA PIP profile of translation-based multiple alignment
    PlotPIPprofiles(
      #    alignments = featureAlignmentsNto1$alignments[seqOrder],
      alignment = featureAlignmentsNto1$tralignedAA, 
      refSeqName = refMatchName,
      plotRefGaps = FALSE,
      reversePlot = TRUE,
      windowSize = windowSize/3,
      main = paste0("AA PIP profiles: collection ", collection,
                    "\nGenomic feature ", featureName, " (", featureStart, ":", featureEnd ,", ", featureLength, " bp)"),
      colors = matchColors,
      legendMargin = 0.3,
      legendCorner = "topright",
      lwd = 1,
      legendCex = 0.5,
      ylim = c(30, 100))
  }    
  
  
}