view R/ltr_utils.R @ 0:7b0bbe7477c4 draft

"planemo upload commit 92c684dff3b377c8c08654c7f3d46a133385e3e0-dirty"

add_coordinates_of_closest_neighbor <- function(gff) {
  gff <- gff[order(seqnames(gff), start(gff))]
  # split to chromosomes:
  gff_parts <- split(gff, seqnames(gff))
  upstreams <- c(sapply(gff_parts, function(x) c(1, head(end(x), -1))))
  downstreams <- c(sapply(gff_parts, function(x) c(start(x)[-1], seqlengths(x)[runValue(seqnames(x[1]))])))
  gff_updated <- unlist(gff_parts)
  gff_updated$upstream_domain <- unlist(upstreams)
  gff_updated$downstream_domain <- unlist(downstreams)
  names(gff_updated) <- NULL
  return(gff_updated)
}

get_domain_clusters <- function(gff) {
  # get consecutive domains from same linage
  # must be sorted!
  gag_plus <- as.numeric(cumsum(gff$Name == "GAG" & strand(gff) == '+'))
  gag_minus <- rev(as.numeric(cumsum(rev(gff$Name == "GAG" & strand(gff) == '-'))))
  # split based on classification - must be same and consecutive
  x <- rle(gff$Final_Classification)
  # split on strand change
  s <- rep(seq_along(runLength(strand(gff))), runLength(strand(gff)))
  domain_cluster <- paste0(rep(seq_along(x$lengths), x$lengths), "_", seqnames(gff),
                           "_", gag_plus, "_", gag_minus, "_", s)
  gff_clusters <- split(as.data.frame(gff), factor(domain_cluster, levels = unique(domain_cluster)))
  gff_clusters
}

clean_domain_clusters <- function(gcl) {
  ## remove clusters wich does not have enough domains or domains
  ## are on different strand
  N_domains <- sapply(gcl, nrow)
  N_unique_domains <- sapply(gcl, function(x)length(unique(x$Name)))
  S <- sapply(gcl, function(x)paste(sort(unique(x$strand)), collapse = " "))
  S_OK <- S %in% c("+", "-")
  min_domains <- 5
  maxlength <- 15000 # max span between domains
  span <- sapply(gcl, function(x)max(x$end) - min(x$start))
  cond1 <- S_OK &
    N_unique_domains == N_domains &
    N_domains >= min_domains &
    span <= maxlength
  return(gcl[cond1])
}

check_ranges <- function(gx, s, offset = OFFSET) {
  # check is range is not out of sequence length
  START <- sapply(gx, function(x)min(x$start)) - offset
  END <- sapply(gx, function(x)max(x$end)) + offset
  MAX <- seqlengths(s)[sapply(gx, function(x)as.character(x$seqnames[1]))]
  good_ranges <- (START > 0) & (END <= MAX)
  return(good_ranges)
}

get_ranges <- function(gx, offset = OFFSET) {
  S <- sapply(gx, function(x)min(x$start))
  E <- sapply(gx, function(x)max(x$end))
  gr <- GRanges(seqnames = sapply(gx, function(x)x$seqnames[1]), IRanges(start = S - offset, end = E + offset))
}

get_ranges_left <- function(gx, offset = OFFSET, offset2 = 300) {
  S <- sapply(gx, function(x)min(x$start))
  max_offset <- S - sapply(gx, function(x)min(x$upstream_domain))
  offset_adjusted <- ifelse(max_offset < offset, max_offset, offset)
  gr <- GRanges(seqnames = sapply(gx, function(x)x$seqnames[1]), IRanges(start = S - offset_adjusted, end = S + offset2))
  return(gr)
}

get_ranges_right <- function(gx, offset = OFFSET, offset2 = 300) {
  E <- sapply(gx, function(x)max(x$end))
  max_offset <- sapply(gx, function(x)max(x$downstream_domain)) - E
  offset_adjusted <- ifelse(max_offset < offset, max_offset, offset)
  gr <- GRanges(seqnames = sapply(gx, function(x)x$seqnames[1]), IRanges(start = E - offset2, end = E + offset_adjusted))
  return(gr)
}

firstTG <- function(ss) {
  x <- matchPattern("TG", ss)
  if (length(x) == 0) {
    return(0)
  }else {
    return(min(start(x)))
  }
}

lastCA <- function(ss) {
  x <- matchPattern("CA", ss)
  if (length(x) == 0) {
    return(0)
  }else {
    return(max(start(x)))
  }
}

trim2TGAC <- function(bl) {
  for (i in 1:nrow(bl)) {
    tg_L <- firstTG(bl$qseq[i])
    tg_R <- firstTG(bl$sseq[i])
    ca_L <- lastCA(bl$qseq[i])
    ca_R <- lastCA(bl$sseq[i])
    e_dist <- bl$length[i] - ca_R
    no_match <- any(tg_L == 0, tg_R == 0, ca_L == 0, ca_R == 0)
    if (!no_match &
      tg_L == tg_R &
      ca_L == ca_R &
      tg_L < 8 &
      e_dist < 8) {
      ## trim alignment
      bl[i,] <- trim_blast_table(bl[i,], tg_L, e_dist - 1)
    }
  }
  return(bl)
}

trim_blast_table <- function(b, T1, T2) {
  b$qstart <- b$qstart + T1
  b$sstart <- b$sstart + T1
  b$qend <- b$qend - T2
  b$send <- b$send - T2
  b$sseq <- substring(b$sseq, T1, b$length - T2)
  b$qseq <- substring(b$qseq, T1, b$length - T2)
  b$length <- nchar(b$sseq)
  return(b)
}

blast_all2all <- function(seqs, db=NULL, ncpus=1, word_size=20, perc_identity=90, max_target_seq = 5000, task = "megablast", additional_params= ""){
  if (ncpus == 1){
    query <- list(seqs)
  }else{
    query <-split(seqs, round(seq(1,ncpus,length.out = length(seqs))))
  }
  if(is.null(db)){
    # search against itself
    db <- seqs
  }
  qf <-tempfile(fileext=paste0("_",1:ncpus,".fasta"))
  outf <-tempfile(fileext=paste0("_",1:ncpus,".csv"))
  dbf <- tempfile()
  script <-  tempfile()
  writeXStringSet(db, dbf)
  mapply(query, qf, FUN = writeXStringSet)
  cols <- "qaccver saccver pident length mismatch gapopen qstart qend sstart send evalue bitscore qlen slen"
  cmd_db <-  paste("makeblastdb -dbtype nucl -in ", dbf)
  cmd_blast <-  paste("blastn -task ", task, " -word_size", word_size,
                    "-outfmt \"6 ", cols, "\" ",
                    "-perc_identity", perc_identity, " -min_raw_gapped_score 500",
                    "-max_target_seqs", max_target_seq, additional_params,
                    "-query", qf, "-db", dbf, "-out", outf,
                    "&"
  )

  # TODO - inspect only forward strand??
  system(cmd_db)
  cmd_all <- paste(paste(cmd_blast,collapse="\n"),"\nwait")
  cat(cmd_all, file = script)
  system(paste("sh ", script))

  bl_list <- lapply(outf, read.table, stringsAsFactors = FALSE, col.names = unlist(strsplit(cols, " ")), sep="\t", comment.char = "")
  bl_table <- do.call(rbind, bl_list)
  unlink(qf)
  #unlink(outf)
  print(outf)
  unlink(dbf)
  unlink(script)
  return(bl_table)
}

identify_conflicts <- function (bl){
  QL <- gsub(".+[|]", "", bl$qaccver)
  SL <- gsub(".+[|]", "", bl$saccver)
  id_with_conflict <- unique(c(bl$qaccver[QL != SL],bl$saccver[QL != SL]))
  id_ok <- setdiff(unique(c(bl$qaccver,bl$saccver)), id_with_conflict)
  single_hit <- sapply(
    sapply(
      split(bl$qaccver, bl$saccver), unique
    ), length) == 1
  id_with_no_hits <- names(single_hit)[single_hit] # except hit to itself
  return(list(
    ok = id_ok,
    conflict = id_with_conflict,
    no_hit = id_with_no_hits)
  )
}


analyze_TE <- function(seqs, ncpus = 10, word_size = 20){
  blt <- blast_all2all(seqs, ncpus = ncpus, word_size = word_size)
  te_conflict_info <- identify_conflicts(blt)
  blt_te_ok <- blast_table_subset(blt, te_conflict_info$ok)
  te_ok_lineages <- split(blt_te_ok,
                                   gsub(
                                     ".+[|]",
                                     "",
                                     blt_te_ok$qaccver))
  gr_representative <- GRangesList(mclapply(te_ok_lineages,
                             FUN = get_representative_ranges,
                             mc.cores = ncpus
  ))
  seqs_representative <- getSeq(seqs, Reduce(c, gr_representative))
  names(seqs_representative) <- seqnames(Reduce(c, gr_representative))
  # TODO - add swithin group verification here, ! exclude self hits!!

  return(
    list(
      seqs_representative = seqs_representative,
      te_conflict_info = te_conflict_info,
      gr_representative = gr_representative,
      blast = blt
    )
  )
}

query_coverage <- function(blt){
  blt <- blt[blt$qaccver != blt$saccver,]
  Q_lengths <-  blt$qlen[!duplicated(blt$qaccver)]
  names(Q_lengths) <- blt$qaccver[!duplicated(blt$qaccver)]
  gr <- GRanges(seqnames = blt$qaccver, seqlengths = Q_lengths,
               IRanges(start = blt$qstart, end = blt$qend))
  return(coverage(gr))
}

multiplicity_of_te <- function(blt){
  # exclude self to self hits and calculate coverage + mean_multiplicity of TE
  # assuption is that TE which are 'identical' to other TE from the same lineage are
  # likely correct
  blt_no_self <- blt[blt$qaccver != blt$saccver,]
  cvr <- query_coverage(blt_no_self)
  L <- sapply(cvr, function(x) sum(width(x)))
  C1 <- sapply(cvr, function(x) sum(as.numeric(runValue(x) >= 1) * runLength(x)))
  multiplicity <- sapply(cvr, function(x) sum(as.numeric(runValue(x)) * runLength(x)))/L
  data.frame(L = L, C1 = C1,  multiplicity = multiplicity )
}

verify_based_on_multiplicity <- function(TE_info, min_coverage=0.99, min_multiplicity=3){
  blt <- TE_info$blast[TE_info$blast$qaccver %in% TE_info$te_conflict_info$ok,]
  mp <- multiplicity_of_te(blt)
  id_ok_mp_verified <- rownames(mp)[mp$C1/mp$L > min_coverage & mp$multiplicity >= min_multiplicity]
  return(list(multiplicity = mp,
              id_ok_mp_verified = id_ok_mp_verified))

}

compare_TE_datasets <- function(Q, S, word_size = 20, min_coverage = 0.95, ncpus=10){
  blt <- blast_all2all(seqs = Q, db = S, ncpus = ncpus, word_size = word_size)
  QL <- gsub(".+[|]", "", blt$qaccver)
  SL <- gsub(".+[|]", "", blt$saccver)
  id_with_conflict <- unique(c(blt$qaccver[QL != SL]))
  id_ok <- setdiff(unique(blt$qaccver), id_with_conflict)
  # check coverage hits
  blt_ok <- blt[blt$qaccver %in% id_ok,]
  Q_lengths <-  blt_ok$qlen[!duplicated(blt_ok$qaccver)]
  names(Q_lengths) <- blt_ok$qaccver[!duplicated(blt_ok$qaccver)]
  gr <- GRanges(seqnames = blt_ok$qaccver, seqlengths = Q_lengths,
               IRanges(start = blt_ok$qstart, end = blt_ok$qend))
  cvr <- coverage(gr)
  L <- sapply(cvr, function(x) sum(width(x)))
  C1 <- sapply(cvr, function(x) sum(as.numeric(runValue(x) >= 1) * runLength(x)))
  Max_uncovered <- sapply(cvr, function(x){
    if(any(runValue(x)==0)){
      return(max(runLength(x)[runValue(x) == 0]))
    }else{
      return(0)
    }
  })

  # verified based on hit to reference - S
  C1_prop <- C1/L
  pass <-  (C1_prop >= min_coverage & Max_uncovered < 500)
  if (any(pass)){
    id_ok_verified <-  names(C1_prop)
  }else {
    id_ok_verified <- NULL
  }
  return(list(id_with_conflict = id_with_conflict,
              id_ok = id_ok,
              id_ok_verified = id_ok_verified
  ))
}



blast_table_subset <- function(bl,id){
  return(bl[bl$qaccver %in% id & bl$saccver %in% id,, drop = FALSE])
}

get_representative_ranges <-  function(bl, min_length = 60){
  score <- sort(unlist(by(bl$bitscore, bl$qaccver, sum, simplify = FALSE)),
               decreasing = TRUE)
  L <-  bl$qlen[!duplicated(bl$qaccver)]
  names(L) <- bl$qaccver[!duplicated(bl$qaccver)]
  gr <- GRanges(seqnames = bl$qaccver,
               IRanges(start = bl$qstart, end = bl$qend),
               seqlengths = L,
               subject = bl$saccver,
               sstart = ifelse(bl$send < bl$sstart, bl$send, bl$sstart),
               send = ifelse(bl$send > bl$sstart, bl$send, bl$sstart))
  SN <-  levels(seqnames(gr))
  inc <- rep(TRUE, length(gr))
  MSK <- GRangesList()
  for (i in names(score)){
    inc[gr$subject %in% i] <- FALSE
    gr_part <- gr[seqnames(gr) %in% i & inc]
    MSK[[i]] <- GRanges(seqnames = factor(gr_part$subject, levels = SN),
                       IRanges(start = gr_part$sstart, end = gr_part$send),
                       seqlengths = L
    )
  }
  gout <- unlist(MSK)

  full_gr <- GRanges(seqnames = factor(SN, levels = SN),
                     IRanges(start = rep(1,length(L)),
                            end = L)
  )
  unmasked_gr <- GenomicRanges::setdiff(full_gr, gout)
  return(unmasked_gr[width(unmasked_gr) >= min_length])
}

expected_diversity <- function(seqs, niter=100, km = 6){
  L <- nchar(seqs)
  R <- matrix(ncol = niter, nrow = length(seqs))
  for (i in 1:niter){
    print(i)
    seqs_rnd <- DNAStringSet(sapply(L, function(n) paste(sample(c("A", "C", "T", "G"), n, replace=TRUE), collapse="")))
    R[,i] <- seq_diversity(seqs_rnd, km = km)$richness
  }
  R

}

seq_diversity <- function (seqs, km=6){
  K <- oligonucleotideFrequency(seqs, width=km)>0
  P <- t(K)/rowSums(K)
  # shannon index
  SI <- apply(P, 2, function(x) {x1 <- x[x>0]; -sum(x1*log(x1))})
  # richness
  R <- rowSums(K)
  list(richness=R, shannon_index=SI)
}



blast <- function(s1, s2) {
  tmp1 <- tempfile()
  tmp2 <- tempfile()
  tmp_out <- tempfile()
  writeXStringSet(DNAStringSet(s1), tmp1)
  writeXStringSet(DNAStringSet(s2), tmp2)
  # alternative blast:
  cmd <- paste("blastn -task blastn -word_size 7 -dust no -gapextend 1 -gapopen 2 -reward 1 -penalty -1",
               " -query ", tmp1, ' -subject ', tmp2, ' -strand plus ',
               '-outfmt "6 qaccver saccver pident length mismatch gapopen qstart qend sstart send evalue bitscore qseq sseq"',
               '  -out', tmp_out)

  system(cmd)
  out_raw <- read.table(tmp_out, as.is = TRUE, sep = "\t",
                        col.names = strsplit("qaccver saccver pident length mismatch gapopen qstart qend sstart send evalue bitscore qseq sseq", split = ' ')[[1]])

  if (nrow(out_raw) == 0) {
    return(out_raw)
  }
  out <- trim2TGAC(out_raw)
  # remove alingment shorted that
  out <- out[out$length > 100,]
  if (nrow(out) == 0) {
    return(out)
  }
  ## filter for TGCA
  TG_L <- substring(out$qseq, 1, 2) == "TG"
  TG_R <- substring(out$sseq, 1, 2) == "TG"
  CA_L <- substring(out$qseq, out$length - 1, out$length) == "CA"
  CA_R <- substring(out$sseq, out$length - 1, out$length) == "CA"
  cond <- TG_L & TG_R & CA_L & CA_R
  out <- out[cond, , drop = FALSE]
  unlink(tmp1)
  unlink(tmp2)
  unlink(tmp_out)
  # TODO - detele all temp files!
  # unlink(tmp1, tmp2, tmp_out)
  return(out)
}

get_correct_coordinates <- function(b) {
  do.call(rbind, strsplit(b$qaccver, split = "_"))
}

evaluate_ltr <- function(GR, GRL, GRR, blast_line, Lseq, Rseq) {
  LTR_L <- makeGRangesFromDataFrame(data.frame(seqnames = seqnames(GR),
                                               start = start(GRL) + blast_line$qstart - 2,
                                               end = start(GRL) + blast_line$qend - 1))
  LTR_R <- makeGRangesFromDataFrame(data.frame(seqnames = seqnames(GR),
                                               start = start(GRR) + blast_line$sstart - 2,
                                               end = start(GRR) + blast_line$send - 1))

  TSD_L <- makeGRangesFromDataFrame(data.frame(seqnames = seqnames(GR),
                                               start = start(GRL) + blast_line$qstart - 3:10,
                                               end = start(GRL) + blast_line$qstart - 3))
  TSD_R <- makeGRangesFromDataFrame(data.frame(seqnames = seqnames(GR),
                                               start = start(GRR) + blast_line$send,
                                               end = start(GRR) + blast_line$send + 0:7))

  TSD_L_seq <- DNAStringSet(substring(Lseq, blast_line$qstart - 2:9, blast_line$qstart - 2))
  TSD_R_seq <- DNAStringSet(substring(Rseq, blast_line$send + 1, blast_line$send + 1:8))

  matching_tsd <- TSD_R_seq == TSD_L_seq
  matching_tsd[1:3] <- FALSE # remove short tsd
  p <- which(matching_tsd)
  if (length(p) > 0) {
    TSD_Length <- max(p)
    TSD_sequence <- TSD_L_seq[TSD_Length]
    TSD_position <- append(TSD_R[TSD_Length], TSD_L[TSD_Length])
  }else {
    TSD_Length <- 0
    TSD_sequence <- ""
    TSD_position <- NA
  }

  TE_Length <- end(LTR_R) - start(LTR_L)
  LTR_Identity <- blast_line$pident
  out <- list(TSD_position = TSD_position, TSD_sequence = TSD_sequence, TSD_Length = TSD_Length,
              LTR_R_position = LTR_R, LTR_L_position = LTR_L, TE_Length = TE_Length, LTR_Identity = LTR_Identity)
  return(out)
}

get_best_ltr <- function(x) {
  tsd_ok <- sapply(x, function(k)k$TSD_Length > 3)
  te_length_ok <- sapply(x, function(k)k$TE_Length < 30000)
  ltr_length_ok <- sapply(x, function(k)width(k$LTR_R_position) >= 100 & width(k$LTR_L_position) >= 100)
  if (sum(tsd_ok & te_length_ok & ltr_length_ok) >= 1) {
    # return the first one (best bitscore)
    return(x[tsd_ok & te_length_ok][1])
  }
  if (any(te_length_ok & ltr_length_ok)) {
    return(x[te_length_ok & ltr_length_ok][1])
  }else {
    return(NULL)
  }
}

get_te_gff3 <- function(g, ID) {
  D <- makeGRangesFromDataFrame(g$domain, keep.extra.columns = TRUE)
  sn <- seqnames(D)[1]
  S <- strand(D)[1]
  TE <- GRanges(seqnames = sn,
                IRanges(start = start(g$ltr_info[[1]]$LTR_L_position),
                        end = end(g$ltr_info[[1]]$LTR_R_position)), strand = S)
  TE$type <- "transposable_element"
  TE$ID <- ID

  if (as.character(S) == "+") {
    LTR_5 <- g$ltr_info[[1]]$LTR_L
    LTR_3 <- g$ltr_info[[1]]$LTR_R
  }else {
    LTR_3 <- g$ltr_info[[1]]$LTR_L
    LTR_5 <- g$ltr_info[[1]]$LTR_R
  }
  LTR_5$LTR <- '5LTR'
  LTR_3$LTR <- '3LTR'
  LTR_5$type <- "long_terminal_repeat"
  LTR_3$type <- "long_terminal_repeat"
  strand(LTR_3) <- S
  strand(LTR_5) <- S
  LTR_3$Parent <- ID
  LTR_5$Parent <- ID
  LTR_3$Final_Classification <- D$Final_Classification[1]
  LTR_5$Final_Classification <- D$Final_Classification[1]
  LTR_5$LTR_Identity <- g$ltr_info[[1]]$LTR_Identity
  LTR_3$LTR_Identity <- g$ltr_info[[1]]$LTR_Identity

  TE$LTR_Identity <- g$ltr_info[[1]]$LTR_Identity
  TE$LTR5_length <- width(LTR_5)
  TE$LTR3_length <- width(LTR_3)

  if (is.na(g$ltr_info[[1]]$TSD_position)[1]) {
    # no TSD found
    TSD <- NULL
    TE$TSD <- 'not_found'
  }else {
    TSD <- g$ltr_info[[1]]$TSD_position
    TSD$type <- "target_site_duplication"
    TSD$Parent <- ID
    TE$TSD <- as.character(g$ltr_info[[1]]$TSD_sequence)
  }

  TE$Final_Classification <- D$Final_Classification[1]

  D$Parent <- ID
  out <- c(TE, LTR_3, LTR_5, D, TSD)
  return(out)
}

get_TE <- function(Lseq, Rseq, domains_gff, GR, GRL, GRR) {
  xx <- blast(Lseq, Rseq)
  if (nrow(xx) == 0) {
    return(NULL)
  }else {
    ltr_tmp <- list()
    for (j in 1:nrow(xx)) {
      ltr_tmp[[j]] <- evaluate_ltr(GR, GRL, GRR, xx[j, , drop = FALSE], Lseq, Rseq)
    }
    ltr <- get_best_ltr(ltr_tmp)
    if (length(ltr) == 0) {
      return(NULL)
      ## add good ltr
    }else {
      return(list(domain = domains_gff, ltr_info = ltr, blast_out = xx))
    }
  }
}

add_pbs <- function(te, s, trna_db) {
  ltr5 <- te[which(te$LTR == "5LTR")]
  STRAND <- as.character(strand(te)[1])
  if (STRAND == "+") {
    pbs_gr <- GRanges(seqnames(ltr5), IRanges(start = end(ltr5) + 1, end = end(ltr5) + 31))
    pbs_s <- reverseComplement(getSeq(s, pbs_gr))
  }else {
    pbs_gr <- GRanges(seqnames(ltr5), IRanges(end = start(ltr5) - 1, start = start(ltr5) - 30))
    pbs_s <- getSeq(s, pbs_gr)
  }

  names(pbs_s) <- "pbs_region"
  # find trna match
  tmp <- tempfile()
  tmp_out <- tempfile()
  writeXStringSet(DNAStringSet(pbs_s), tmp)
  # alternative blast:
  cmd <- paste("blastn -task blastn -word_size 7 -dust no -perc_identity 100",
               " -query ", tmp, ' -db ', trna_db, ' -strand plus ',
               '-outfmt "6 qaccver saccver pident length mismatch gapopen qstart qend sstart send evalue bitscore qseq sseq"',
               '  -out', tmp_out)

  system(cmd)
  pbs_exact_gr <- NULL
  out_raw <- read.table(tmp_out, as.is = TRUE, sep = "\t",
                        col.names = strsplit(
                          "qaccver saccver pident length mismatch gapopen qstart qend sstart send evalue bitscore qseq sseq",
                          split = ' ')[[1]])
  if (nrow(out_raw) > 0) {
    cca <- grepl("CCA$", out_raw$qseq)
    to_end <- out_raw$send == 23 # align to end of sequence
    max_dist <- out_raw$qend > 25 # max 5 bp from ltr
    min_length <- out_raw$length >= 10
    out_pass <- out_raw[cca & to_end & max_dist & min_length,]
    if (nrow(out_pass) > 0) {
      trna_id <- out_pass$saccver[1]
      if (STRAND == "+") {
        S <- end(ltr5) + 32 - out_pass$qend[1]
        E <- end(ltr5) + 32 - out_pass$qstart[1]
      }else {
        S <- start(ltr5) - 31 + out_pass$qstart[1]
        E <- start(ltr5) - 31 + out_pass$qend[1]
      }
      pbs_exact_gr <- GRanges(seqnames(ltr5), IRanges(start = S, end = E))
      pbs_exact_gr$trna_id <- trna_id
      pbs_exact_gr$Length <- out_pass$Length
      strand(pbs_exact_gr) <- STRAND
      pbs_exact_gr$type <- 'primer_binding_site'
      pbs_exact_gr$Parent <- te[1]$ID
      te$trna_id <- c(trna_id, rep(NA, length(te) - 1))

    }
  }
  te <- append(te, pbs_exact_gr)
  unlink(tmp)
  unlink(tmp_out)
  return(te)
}

get_te_statistics <- function(gr, RT) {
  DOMAINS_LTR <- gr[gr$type == "transposable_element" &
                      gr$TSD == "not_found" &
                      is.na(gr$trna_id)]
  DOMAINS_LTR_TSD <- gr[gr$type == "transposable_element" &
                          gr$TSD != "not_found" &
                          is.na(gr$trna_id)]
  DOMAINS_LTR_TSD_PBS <- gr[gr$type == "transposable_element" &
                              gr$TSD != "not_found" &
                              !is.na(gr$trna_id)]
  DOMAINS_LTR_PBS <- gr[gr$type == "transposable_element" &
                          gr$TSD == "not_found" &
                          !is.na(gr$trna_id)]

  all_class <- names(sort(table(RT$Final_Classification), decreasing = TRUE))
  RT_domain <- as.integer(table(factor(RT$Final_Classification, levels = all_class)))
  DL <- as.integer(table(factor(DOMAINS_LTR$Final_Classification, levels = all_class)))
  DLT <- as.integer(table(factor(DOMAINS_LTR_TSD$Final_Classification, levels = all_class)))
  DLTP <- as.integer(table(factor(DOMAINS_LTR_TSD_PBS$Final_Classification, levels = all_class)))
  DLP <- as.integer(table(factor(DOMAINS_LTR_PBS$Final_Classification, levels = all_class)))
  out <- data.frame(RT_domain = RT_domain,
                    DOMAINS_LTR = DL,
                    DOMAINS_LTR_TSD = DLT,
                    DOMAINS_LTR_PBS = DLP,
                    DOMAINS_LTR_TSD_PBS = DLTP,
                    row.names = all_class
  )
  total <- colSums(out)
  out <- rbind(out, Total = total)
  return(out)
}

getSeqNamed <- function(s, gr) {
  spart <- getSeq(s, gr)
  id1 <- paste0(seqnames(gr), '_', start(gr), "_", end(gr))
  id2 <- gr$Final_Classification
  names(spart) <- paste0(id1, "#", id2)
  spart
}

get_TE_id <- function (gr){
  gr_te <- gr[gr$type == "transposable_element"]
  ID <- gr_te$ID
  A <- paste0(seqnames(gr_te), '_', start(gr_te), "_", end(gr_te))
  B <- gr_te$Final_Classification
  names(ID) <- paste0(A, "#", B)

}

get_te_sequences <- function(gr, s) {
  # return list of biostrings
  DOMAINS_LTR <- gr[gr$type == "transposable_element" &
                      gr$TSD == "not_found" &
                      is.na(gr$trna_id)]
  DOMAINS_LTR_TSD <- gr[gr$type == "transposable_element" &
                          gr$TSD != "not_found" &
                          is.na(gr$trna_id)]
  DOMAINS_LTR_TSD_PBS <- gr[gr$type == "transposable_element" &
                              gr$TSD != "not_found" &
                              !is.na(gr$trna_id)]
  DOMAINS_LTR_PBS <- gr[gr$type == "transposable_element" &
                          gr$TSD == "not_found" &
                          !is.na(gr$trna_id)]
  s_DL <- getSeqNamed(s, DOMAINS_LTR)
  s_DLT <- getSeqNamed(s, DOMAINS_LTR_TSD)
  s_DLP <- getSeqNamed(s, DOMAINS_LTR_PBS)
  s_DLTP <- getSeqNamed(s, DOMAINS_LTR_TSD_PBS)
  return(DNAStringSetList(
    DL = s_DL,
    DLT = s_DLT,
    DLP = s_DLP,
    DLTP = s_DLTP
  ))

}

cd_hit_est <- function(seqs, min_identity = 0.9, word_size = 10, ncpu = 2){
  # runs cd-hi-est and return table with cluster membership, and size and if reads was repesentative
  # input sequences must be in the same orientation!!!
  sfile <- tempfile()
  fasta_out <- tempfile()
  clstr <- paste0(fasta_out,".clstr")
  # cdhit is triming names!!
  ori_names <-  names(seqs)
  names(seqs) <- seq_along(seqs)
  writeXStringSet(seqs, sfile)
  cmd <- paste("cd-hit-est",
               "-i", sfile,
               "-o", fasta_out,
               "-c", min_identity,
               "-n", word_size,
               "-T", ncpu,
               "-r", 0)
  system(cmd)
  cls_raw <-  grep("^>", readLines(clstr), invert = TRUE, value = TRUE)
  unlink(fasta_out)
  unlink(clstr)
  index <-  gsub("\t.+","",cls_raw)
  id <-  as.numeric(gsub("[.].+","",
                       gsub(".+>", "", cls_raw))
  )
  is_representative <-  id %in% id[grep("[*]$",cls_raw)]
  membership <-  cumsum(index=="0")
  cluster_size <-  tabulate(membership)[membership]
  # reorder
  ord <- order(id)
    cls_info <- data.frame(
      seq_id = ori_names,
      membership = membership[ord],
      cluster_size = cluster_size[ord],
      is_representative = is_representative[ord]
    )
  return(cls_info)
}