GOslim Counts - P.evermanni Expressed Genes and BLASTing to GOslim for deep-dive-expression Project

2025
E5
deep-dive-expression
GOslim
Porites evermanni
Author

Sam White

Published

June 20, 2025

INTRO

This notebook annotated expressed genes with gene ontologies, assigned them to GOslims, and generated corresponding counts of GOs in each GOslim category, as part of urol-e5/deep-dive-expression (GitHub repo).

Note

The contents below are from markdown knitted from 30.00-Peve-transcriptome-GOslims.md (commit 0b980cb).

1 BACKGROUND

This notebook will perform annotation of expressed genes, as previously determined by 06.2-Peve-Hisat.qmd (GitHub).

Briefly, the notebook will perform the following tasks:

  1. Extract all genes from the genome, as GFF and FastA.

  2. Create a subset of only expressed genes, based on gene count matrix.

  3. BLASTx expressed genes against SwissProt database.

  4. Get gene ontology.

  5. Map gene ontology to GOslims and get counts.

Important

Expressed genes were defined as those genes having at least one count across all samples.

1.1 INPUTS

  • Gene count matrix

  • Genome FastA

  • Genome GFF

1.2 OUTPUTS

  • Genes BED

  • Genes FastA

  • Expressed genes FastA

  • Expressed genes SwissProt IDs only file.

  • Expressed genes to SwissProt IDs mapping file.

  • Expressed genes to SwissProtIDs and GO mapping file.

  • Counts file of expressed genes GOslims.

1.3 SOFTWARE

2 VARIABLES

# DIRECTORIES
top_output_dir <- file.path("..", "output")
output_dir <- file.path(top_output_dir, "30.00-Peve-transcriptome-GOslims")
data_dir <- file.path("..", "data")

# PROGRAMS
blastdbs_dir <- file.path("..", "..", "M-multi-species", "data", "blastdbs")
programs_dir <- file.path("", "home", "shared")
bedtools_dir <- file.path(programs_dir, "bedtools-v2.30.0", "bin")
blast_dir <- file.path(programs_dir, "ncbi-blast-2.15.0+", "bin")
diamond <- file.path(programs_dir, "diamond-2.1.8")
fastaFromBed <- file.path(bedtools_dir, "fastaFromBed")
samtools_dir <- file.path(programs_dir, "samtools-1.12")
samtools <- file.path(samtools_dir, "samtools")



# FILES
count_matrix <- file.path(top_output_dir, "06.2-Peve-Hisat/gene_count_matrix.csv")
diamond_db <- "20250620-diamond"
diamond_output <- file.path(output_dir, "Pevermanni-expressed-genes.blastx.outfmt6")
genome_fasta <- file.path(data_dir, "Porites_evermanni_v1.fa")
genes_fasta <- file.path(output_dir, "Pevermanni-genes.fasta")
genes_fasta_index <- file.path(output_dir, "Pevermanni-genes.fasta.fai")
genes_subset_fasta <- file.path(output_dir, "Pevermanni-subset-genes.fasta")
genes_subset_fasta_index <- file.path(output_dir, "Pevermanni-subset-genes.fasta.fai")
genes_bed <- file.path(output_dir, "Pevermanni-genes.bed")
og_genome_gff <- file.path(data_dir, "Porites_evermanni_validated.gff3")

# THREADS
threads <- "40"

##### Official GO info - no need to change #####
goslims_obo <- "goslim_generic.obo"
goslims_url <- "http://current.geneontology.org/ontology/subsets/goslim_generic.obo"

# FORMATTING
line <- "-----------------------------------------------"

# Export these as environment variables for bash chunks.
Sys.setenv(
  blastdbs_dir = blastdbs_dir,
  count_matrix = count_matrix,
  data_dir = data_dir,
  diamond = diamond,
  diamond_db = diamond_db,
  diamond_output = diamond_output,
  fastaFromBed = fastaFromBed,
  genes_bed = genes_bed,
  genes_fasta = genes_fasta,
  genes_fasta_index = genes_fasta_index,
  genes_subset_fasta = genes_subset_fasta,
  genes_subset_fasta_index = genes_subset_fasta_index,
  genome_fasta = genome_fasta,
  og_genome_gff = og_genome_gff,
  output_dir = output_dir,
  top_output_dir = top_output_dir,
  line = line,
  samtools = samtools,
  threads = threads
)

3 Extract genes as FastA

3.1 Extract genes from GFF

# Read GFF, skipping comment lines
genome_gff <- readr::read_tsv(
  og_genome_gff,
  comment = "#",
  col_names = c("seqid", "source", "type", "start", "end", "score", "strand", "phase", "attributes")
)

# Filter for gene features
genes <- as.data.frame(genome_gff) %>%
  filter(type == "gene") %>%
  mutate(
    chrom = seqid,
    start = start - 1,  # BED is 0-based
    end = end,
    gene_id = sub("ID=([^;]+);?.*", "\\1", attributes)
  ) %>%
  dplyr::select(chrom, start, end, gene_id)

str(genes)
'data.frame':   40389 obs. of  4 variables:
 $ chrom  : chr  "Porites_evermani_scaffold_1" "Porites_evermani_scaffold_1" "Porites_evermani_scaffold_1" "Porites_evermani_scaffold_1" ...
 $ start  : num  3106 8491 26133 30312 32615 ...
 $ end    : num  4488 11026 26404 42225 67628 ...
 $ gene_id: chr  "gene-Peve_00000001" "gene-Peve_00000039" "gene-Peve_00000112" "gene-Peve_00000117" ...

3.1.1 Write genes to BED

# Write to BED
write_tsv(genes, genes_bed, col_names = FALSE)

3.2 Create genes FastA


"${fastaFromBed}" -fi "${genome_fasta}" -bed "${genes_bed}" -nameOnly > "${genes_fasta}"

# Create FastA index
"${samtools}" faidx "${genes_fasta}"

head "${genes_fasta}"

echo ""
echo ""

head "${genes_fasta_index}"
>gene-Peve_00000001
TTACTGCTTCAGTATGTGAATTTCGATGGTGGCTTGACCGGAGTTAGACATGGCCCCCCTTGCTCGGAGTCCCGATCCCAAATCTCTCTCGTGCCAGTAGTTATCTCCTTTGAAGGGATTATCGTAATACATCCGGTACCAAAGATTGTAGTTGGCGCGTCTTTTACCACTGTAAAGCCAAACATCAAACCAGTTGCTGTACCAGTTGTAGTCAAATGGAACGGAATACATAACAGCAAGTGTTTTATCGATGTGTGGGATGTAGTATGTTAATACTCCTACTGCGCCTCTCGCAACGGGCCCCGCAGTTTTTCGTGCGCCGTAAAGCAAAGCTGTGCCTAGAAATAAGCAAACAAATTAGCAATTAAACAAAAATCATAAACATGATCATTCGTCACAATGATACATGGCAAACNTTGTGTCAGGTTAGTGATTGCTTATCTTTTCCCCAAATATTACACGCAAAGGGATTCTCATTCTGAACATGAATACTAAGTTTAGTTGGTTTAAGAAGCTCTTTCAACCCTGAAGTCATTTTGCCGTTCTGACAAAGCAAAACATAAACCTGTTGTCTGCAAAATTTCAAGAATGGGCAAATCCTCTAATACAGTTTCTTTACTCTTTAGGGTAAGTGGTCGCTGAGTTCGTCCTTGGTTTCATACGGTAATAGGCCCTTTGCACGCGATGAACTCATTTTACTACTACTACTACTACCAGAATCACTCGGGGTTTTGCTTTCTTGGACAAACGAGGGCTTTATTTTTCAAACCTCGCATAACCTGTGATCAGGCCCCCAAAACAAAAAGGGAAGAAGGACCGCCTGATTGCAGGTTAAATTCTTCGCAGAGATTAGCTAGAAAAATTAAATATGACAAGAAAACCAAAATGGATTCTATAGCAATAGATCAGACCTTTTTAGCTTGTATGGTTTGTTTACCCATTTCAGACCAAGTGATGGTACCCCAGAGAACTGTTTCTTTCAATGTCGTCTTTTGCACGTGCACCCACATGTTATGTATGCATAATTATATAAAAGACAAATTCCCCCAAGAACATCACGTGGTCTGAATTGGGAAAACAAAACAAACAAGCTAATGACACCATCCTGCATACTGGCCTTAGTGTTATTACTAAAAGAAAGGATTTTAAACTTTTATGTTATTAATATTAATTTTACCTGAAGAAACATCATGAGGCAAAACACGATTTGACGTCCCTGAATAAAAATATATGTTGACTGCTCTCCATTTATATCCACTTTCGTTATCAACACCAATGGCGACCTTGCGGCTTATGCTACCAAGGGTGTTTAAAATTGTTGTGAGAATGCCCAAGCCAAGTTGAGCACCGCTGATGACAGCACCAGCGTCAGCTAAAATTTT
>gene-Peve_00000039
ATGGGCATCGTTAGGCTGTTCACCCTTCTGCTTTTCGTTCTGCTGATAACTCAGTCAATCTTTGGGTCTCCCTTCAAGCAAGACAATCATGTCGAAAGGAGTCCTGGATCGTCCTCTAAACACACTGAGGTAAAACAGTTCTCTTTAAAATAAGATAGTCAATTGGTGTCTTGTGTTTAAGACGAACATGAAAGTGTCTCTTGGGGGTGGCGTGATATCTACGGAAATGCAACTCACTGTTCTAATCGGATCTCGGTTGTTTCACTAAAACAAAATGAACCCTAATCTCGGACCGAATATAAAACCGACTAACACCCCCCCCCCCCCCCTCCAGTTCAGAATTCTAATCTCGACACTCGCTTTCCCTTTTCTCCAAAACACCGATTACCCTTGTGAGTGTGTAGTGCTGCGAGTAGATACTAGGTAGTTTTACACCTTAACGCTTGAATGTATAGTAAACATTTTAATAATTTTAAGCCGTAAATCTATTGAGTTCTTTGTGATGTGTCCCTCAATAATCAATGTTCAGTTTGGCGAAGTGTAAACTGAATGAATACACAGCCATCATGCAATTCTTGGTGGTTTCGCTAAGGCTCCCAGCGATAATTTTAGCCGGCTTCCGATTTCATTTCTGTGCCTCAAATGTATTTAAATACACAGAGGAAAAATTAGTGAAAAAAAAAGAATACTCATTCCAGTTTGTATTCAGTGGAATGACCTTAATGGCCATACTTTCCTTCACAGATACAGGGACGACAAGGCTCGTCCACGATCTCTACTCGTAAAGAAGGCGACGAGAAAAACAAGCGAAAAGGTAATTGCATCAGTTGTTTAGAAGTTGCGTACCATAACCTTTGAAAACAAAAGGAAGAAACAAAAAGAAGCTGAAACGTAACTGCACAGGCTAGCTGTTTGGTAAACCAAACTATTCATTACAGTTGTTGGGTTACTCTGCTAAACGAAAACATTCATTAGCCGACTGTTTATTCAAACTTGGTTTGTTTTAATACCACTAAGTGTGCAACTAAAAATTTTATCTTGCTGTGTTCTTTAAAATTTTAGCTGACGCTGGTGCTGTCATCAGCGGTGCTCAACTTGGTTTGGGCATTCTCACAACAATTTTAAACACCCTTGGTAGCATAAGCCGCAAGGTCGCCATTGGTGTTGATAACGAAAGTGGATATAAATGGAGAGCAATCAACATATATTTTTATTCAGGGACGTCAAATCGTGTTTTGCCTCATGATGTTTCTTCAGGTAAAATTAATATTAATAACATAAAAGTTTAAAATCCTTTCTTTTAGTAATGACACTGAGGCCCGTATGCAGGATGATGTCATTAGCTTGTTTGATTTGTTTTCCCAACGTTCTTCGGGGAATTTGTCTTTTATATAATTATGCATACATATACACGTGGGTGCACATGCAAAAGATCAGACGACATTGAAAGAAACAGTTCTCTGGGGTACTATCACCTGGTCTGAAATGGGAAAACAAAACGTACCAGCTAAAAAGGTCTGATCTATTAGTATAGAATCCATTTCGGTTTTCTTGTCATATTTAATTTTTCTAGCTAATCTCTGCGAAGAGTTTAACCTGTAATCAGGCGGTCCTTCTTCCCTTTTTGTTTTTGGGGGCCTGACCGCAGGTTATGCCAGGTTTGAAAAGTAAAGCCCTCGTTGGTCCAAGAAAGCAAAACCCCGAGTGATTCTGGTAGTAGTAGTAGTAGTGAAATGAGCTCATCGCGTGCAAAGGGCCTATTACTGTATGAAACCAGGGGCGAACTCAGCGACCACTTACCCTAAAGAGGAAAGAAACTGTATCAGAGGATTTGCCCCTTCTTGAAATTTTGCTGACAACAGGTTTACGTTTTGCTTTGTCAGAACGGCAAAATGACTTCAGGGTTGAAAGAGCTTCTTAAACCAACCAAACGTAGTATTCTGAGTGTGTGATGTTCAGAATGAACATCCCTTTGCGTGTAATATTTGGGGAAAAGATAAGCAATCACTAACCTGACACAAATAAAATTTCTGGGTGGCAACAAAGGAGGACATGTTAATGATCTGACACATTCTATGTAAATTTTGTTTTTGGGTACACGACTCTATTCTGTGATGCTTGTTTGCCATGTATTATTGTGACGAATGATCATGTTTATGATTTTTGTTTAATTGCTAATTTGTTTGCTTATTTCTAGGCACAGCTTTGCTTTACGGCGCACGAAAAACTGCGGGGCCCGTTGCGAGAGGCGCAGTAGGAGTATTAACATACTACATCCCACACATCGATAAAACACTTGCTGTTATGTATTCCGTTCCATTTGACTACAACTGGTACAGCAACTGGTTTGATGTTTGGCTTTACAGTGGTAAAAGACGCGCCAACTACAATCTTTGGTACCGGATGTATTACGATAATCCCTTCAAAGGAGATAACTACTGGCACGAGAGAGATTTGGGATCGGGACTCCGAGCAAGGGGGGCCATGTCTAACTCCGGTCAAGCCACCATCGAAATTCACATACTGAAGCAGTAA
>gene-Peve_00000112
TTCTGCTCTCTATGTTTAGGTCAGCCTTATCAGCGTGGTCTCACAGCTATTCTTTGCACGATAGGCAGTGATAGCATCACTAAGTCTGTCATCCATAAACTTACTGTCTTGTTGGCCCAACAGTACTTCATAAACTTTATTCACGCATGGCAACACCGTGATAGGTCTATAGTTTATATCCCATTGTTTATCCAGTTTATCATCCTTCTTATGTACTGGTGTCCATTCCCCACGCTTAAGCTTTGCTATCCATTCTCCATTTCTAATCGCT
>gene-Peve_00000117
AACTTTCTTGCTGCAGTGAAGGACCTGAAGATACTAAAAACTCTGAGGTGAGTACCCCAGTAATGATTGTATTTCGTACGAAAAAAGTTAACAGGTCTTTGGCTAAAGAAAATAGATTTCTAAAATAATATTCTTGTTTTTAATAACGAAATTTCCACTGAAAGCACTCATTTTGCGACAGGTTTTAGATCCTTTGAGCTTTATGTTTAGCTCAGTGTTGCACACTTTTATGTTCGATTCGATTTTATGTTCAATAAACAGCCTGTCCGGCACTTTAAAAAAAAACTGAATAAGACTTGCTAAAATTTCTTTCATGTTTAAACAATCAATTGAAACTCTTAGGACTATTCTTGTTTTGCCTTCATTAAGCCTGTTCAATTCACTTGGCTGTTCATCATGTCAAAACCAAATTTTGGCGTTACTTTATCACCTATGCAGCTCAACCTTTCCTTGGTTTTTCTTTCGATTAAGTTTCTCGTTCTTTACCTAAAGCTGAAAAATCGAAAAAACCTCCAAACCAGTGCAACACTGACAACTTTTCCTGAATTTTTAATTGAGGAATAAAGCTTTTAATTAACTCCAACGTCAGCTTAGTAAGGTGCTTCCTTTTCAGACACAATCGTGAGGGAGAGGAATAATTTTGGGGAAGTTTCCATTAATAACTGTTCGCCAGAAAATTACTTTAAGATAAAAACAACTGAGTTCATGTTTTTTAAGACTATAATTTTAATGGGAAACCTGTGTTGATGCGAGAGTTTCAAAATTTTTCGTATTTATGGCATGCTTTTTGCATCAACATTTTCGCAGTTTCAATAACCCTGTCAAACAATACACACTTTTTGTTTCTTTGGCAATGTTTGTAAACCTCCTGGTCTTCTCAAACTTAACTCGAGAAGGAAAATTAACCGGGTTATGTTTAATAAAGCTTTTGCTTCACGCAAAAATGAAATTTCGGCACCCCATGTTTTCACTTGAATTTAAGTAGTTACATAAGCTTAGCTAAACTTTCATTTCCGCAAATCAATAAAACAGCGTTAACGTTGTGCGGCAAACTTTGCCCCTAGAATTTCTATCAGTGCTTTTTCCTTTCTTGTATAAATATTTTAGGTTTTTAATTGCCTTTAAGATTCAGAATGGAGCACACTGTATTTGTCATCTTTGTTTTGATCCTTGGAATAACTGTCACCACCTTGGGCTCTCCGATGTCAGACGATCATAAAGACGGCGATCCTGAACTATCTCCAAGGCTTAAGGTATACAAAAACGTTTTTTAAGGAGTTATTTTGCTCGAAATTCCTGTGTGGAAGCTGCATTTTTTTTATTCATCAGAGACCTCTGGCACGGCCTCTGAGCAGTACTGTGGCACAGCTGTGTAGCCGTCTCGTTACCCTTTCAGCAGTTAGTTAAGCTAATTCAATGGTTACTTGTTGTTTTTTGTTTTTGTTTTTTATCAAAATGATAGAATTTTGTTTGATCAGTCTGTATCGCCCATAATGCACCACGAAAAGCATAGTCATTCTTTAACGACAGGAAAAGGGACATTGTTTTTGTTTCGCAAAACAAATAAAAAACGAAAAGTGTGTTTTAACTGTTATTTACCGTTCTCTGAGCCTTCCCCAAATTCCAAAGTTTAGATAAGTGATTCTGCACAATAATTGTTCTAGCTGGTCTAGTGTAGCCGGAAACACTGGGAAAGTTAAATTAATTGGTGCTGTTGCCATTTCATTATTTTGTTTTGCATCTGGCTTGTTCATTTAAACTTTCCGCTAATTCTTATTTTTCCACACTGGTCATCCCATTCAGAAGTATCTCAAATAGGCTATTTTTCAAGGAAGGAGAAGGACAACTGGGGAAATTTAACTTAGAAATGACAAAAGAATTAGGTTAAAAACATGTATATTGACTTTAATTAAAAACCACAAATGTTTCTGTTTGATATAAAGAAAATTGAAGGACTGAGAATGCGTTCAAATCAATCAAAAGTCGCATTGGCAAAGGATGCTGAAAAATCAACAAAAGTCCAGGTGAAGTTATTGTTAGCCGTTCTATTTCTCATTTAATCATACGAGTGATTAACAAAATCGGACGACCGCGTAGCGGGAGTCCGATTTGTTTAATCACAAGTATGATTACAGACCGAATTGGATGACACGAAGTTCTGTTACCAATTAATCATAACTTTAACAAAATTTGTGATATATAGGGCTCTTTTTAAAATCAAAACCAAAGAAATTCCAAGATTTTTTCGCTAGCAATGAAAAAAAAGCCATTTAAGAGCGCGCGTGATGGCGCACACTGTCCAATTACTTATGCATGACGCGTACTGTCCTATTAAACTGTCCTATTAAGGCTGAAATCAGGGCAGTTGAGAACCAATCAGATTTGAGAATTTTGTTATAGTTATGATTACAGACCGAATTGGACTCCACTCAGTCCTGTTACCATTACATACTACTGAGCATATACACACGGGAAAACGCGATACTTATCTTAAACGAAAATTCACGGGTAGCCTGCGAGGCATGTTAATACTATCGATGCATTGGTACCGGAGAAAGAGAAATTCAAGTTAGTTCCAAGGGAAAATATTTCACCCTTCAATGTCAGCGTCTTTGGTTGTCGCCAGCACTACCACTATGGTATAAAGAGAGATCGTATAAAAAAAATAAACTTTGCTTGAACATATTTATCTCGACAGGAACGAAAAGAGCAAAGGACCAAGCGTCAAGAACCAGGTCAGTAGTTCATGTATGACCCCATGAAATGTCCTGAGACACCAGTGCCGTGTCGGGTCTCTAGGCACAGAAGCAAGTGTCCAAGAACCAACACGTAATATACCTTTCATTTCCTTCTTTATTGTGTAACATAGGTCAGGTGTTCATACGTTAATTAATTATTTATTTATTTATTGAGAAGCAATAACAGACCCCATGCAAAGGTCTGCTCCCAAAATACAAAAGAGATCCTCAAAATTTTTTTCGAAATTGTATGGCTTAAGACATCTCGAAGAGTCTTTCGTGTTGGAGCCCAGTATGGACCCAGTGGAGGCTGCGAGTTTATATATTATAAATTTGCCGCAAGTGGAAGGTATCTTGAACATATGCCATGCATTTGTAGGGACCGCGGAGCAGGGTTTGGAGTTGTGGGGGGAGGGGGCAGTTAATCTGAACCCGTAAGGGTGAGCCTACTAGGGGATACAGGGGCATGTTTCCCCTGTAAAATTTGATTTTAGGAGTCCTGAAACGGCTAGAAAAGCACCTAAAACTATTATACTTAAATAAAAGTAACGCAAGGCCAAATTTTTAACGATCATATCGCTGCTAAAGTCTGTGAAAGTTGTAACCGAGCTGGTGCTCCTTCAACTGGAAAAGGCAAGATAATTAAAAGGTATAATCGTACCAGTCTCACAATATTTAGGCAGTAAAATAAATCCAACTCCATCAATGACTTGTGCCTTGTTCGAAGTTGTTGCGGATTGGCCGCTTCATCTTCTGACAACTGAGTATCTGTCATTATTGGCTCAAACCTACCACACCACGCTGTTGAATATTTTTAAAAGACGTTTTCTGAACGTTCTTCCCTTTTTATCCCTACCAAAGCCTCTCGTCAGGTTTCGATCAGGTTCCTCATTGCGTGTTTCCAACTTGAAGTTTAAAACAGCATTGATAGAAGAAAGAAGATTCAATAACAAAACCTTCGTAACGTCCCAGTGTAACAAAACCAAACATTCAGGCCTCCTGTTGTTCAATAGGTGGTTAGCGCTACTCTCTCCTCCGCAGAGGCGTCTTTGTGTCGTAGGGAGGCTGGGGAGAAAGAAATAGAAAGCGCGCGGGGCACGGTGGGAAGGGGAGAGAGAGAAAAGAGGCTCCCGTCTTTTCCCTCTTCCCATCTCCCCCCCCCCTCCCCCCCTCGCACTTTTTATTTTTCGATTATTGCTATTTTTATTGTGATACCCAGCAAGAGCCTCTGCGGAGGAGAGAGTTAGCGCTAGCCGACGGATAAATCACTATCCAGTGGATAAGTATTAGGGGTGAATCATTTGAGCTATCCGCTAGGTAAAGATTTATCCGAGCGCTATCCACCTTTTGAACTACTGAAGCTGGCGGTAAAACATTGATCCAAAGGCAAATTAATTCTGGAAAGAAACAGAAGTTGTTAACTCCATTATTTTACCGTCAAAAATCCAAGCGTCCTTTCAGCAACAATAAGCCTTATCACGGTTGTCGACGACATCTTTTGAAAAGTGGGCAACGGCGTGAGAAATTACAGTGTTTGTATGAGAATCTAATATCGAATAATTTCCCTAAAACGGCGGTTCTGAAAAAGATATGAAGTGTAAACTAAAGCTTTGACTCCTAACGATTCTACTGAAAAAATGTAAGGGCGTAACATGCAGTTACATTCGCTAGAACCACTTTATAAAATTTTCTATACATTTGTCTGTAAAAATTTCTCTTCCGACTAAAATTTTCCGGGAAAAATTGCAGACACCGTGAATATATGGAAAACCTAAAGCAAGCGTTCTGGAGAAATTGAAGCACAGTAACAGCCCCCAAAATTTGTTAGTAAAATCCTTTCCTGTGTAGCTTTATTTAATTCATATATCTTTTTTCAGAACACCCGTTTTACGGAAATTATTCGACATTAGATTCTTCTACAATCACTAATTTCTCACGCGGTTGCCTACTCGTCAAAATGGCGTCGAAAACCGTGATGAGGCCTGGAAGGAACAAAAAGAAAAAACAATAGTATTGCAAAGTTTACAAACCAGAGACCAGATTCCGGACAGTTATGTGTTATCACAGCATGGAATTTTTGTCACTGCAGGCGCAGACGTCCCTCCTGGCGAGACGTCCCTTGCGGCGAGGAGCGAGGAGGGAAGGCTGTATTCGCAGGCTAGCGATTGTGGTGATCGTATAGAAAAAATGCACACCCCTAAACGTTTTTGTTTGGCTTTTTTCACACATAGCTGACATAATCGCAGCGGCTGCACTAACTGTTGGCGTACTTCAAGGGATCTTGGATGGCATCATAGGTGTAGATCGCAAGATAGCCATCGGCATCAAGAATAGAAGCGGATATAGCTGGGGAGCAATTGATATTTACTTGCAACAAGGAGTTACAGATAGGGTCATTCCCCGTAGAGTCGATAATGGTAACTATGAATGTCACGAATTTAGCATGAAAGCGCTTATCGGGGACACTGTTATAATGTCTCCTTTAGGAGACGGGGTGGCCATTTCTACGTAGTCTTCGCCCAATTTGATTAATCACTTTTTGTGCTGGAAGAGAAACAAGTCAGTACGTGCCCCTCCTTCAAATTTTTTTCCATTACTTTAAGTTTTTGTTATGGTTTTAGCTTCTCCCTCTTCTTCTTACTTAGGATTTGAGCGTAACCCATTAAGGTAGCTCAAGCTCGAAATATAAATTTGACCAGAGCGCCGTTGTGTCGAAATATGAGGATTTGTGTGGGAAAACTGATATAGCTTTTTAAGGGGGAAATTCGGCTGTTTGTTTTAGGGGGGACATGGGGCTGATTTGAGAAGGAAAGAAATAAGGGAAGAAAAGGTATACACATAAATGACCAGTTAGTTTGGCATTTGTCTACTAATTATTCTCTTCATATTTTAATTATTAACCTCCCCAAATTCAATTTTCCATTATTTTAAACATTTTCCCCCAAATTTTATTCATCAAAACCATCCAAAAACAGTCGCACATTGAGCCAACGACAACGACCTTATTGAGCCTTTTATTATTCAATGAAGTCTTGTCTAGATTTGGAAGAAAAAGAGCAGAAATAACGAAAAACAATGCTGTTGAGTAGAATTCCCCCTTTACTAAAACTGTAAACCAGTATTGAACAAAAACTTAACCAGGTCTAGTTCTCTTTTTGTTTGCCATTTTGCCTGATGTGTGGTTTATTGGTGGCCTTTGGTCCACTCCGAGTTTTGGTTTATATACCAACCCTTTAAAATTAAAGACCCAAAGGTCAGGGGCACCCAACGAGAATATAGTTCAAAACCACTTAACATAGCATTTTTGAACGTAATTTAGTCTTTAAACTGTAGATATAAACATATTTTTATCCCCTAACAATTTTTCATCTGTTCGGATTTCCTAGGTGAAAATCTAGGGATCCGAAAATTATAGGGATCAAAACTTAACTTTTCGAAAATTTCAGTCAGAAAAAAGGCTCCCGAAAATTCTAGATGACCTTTTTAGGGTAAAAATCCGCTTAAAATGGGCAATTGTAGCATTTTTTACATGTTCGAAAATCCTAGGAGAGGCAGGCAAGCAAGAAATTTTATAACAAATGTTCCGAAACTTCTAGATCTCAAATCGTCTTCCGAACAGATATTTTCCGAAAATTGACGTTGGGTGCCCCTGAAAAGTCGATAAGAATTCACGCAGGACGAAAAGAAACAAAGAACTAAGCGAGGTAGAACCGAGGTAGGTTATTTCACAATGCAACACTGTTGCCCAAATGTTAACCATATCGGCTTTTCCCCTACAAAACTCCATATTTCGGCTCATATCTCGAGCTTGGGAAAAAACCTCTTCGACAAGTTATGATGTTGTTTTCATGATGTTGATGATAAGTTCATTCAATATCGTAAATGCTCTTGGTTTTTGGCCATCTCAGTTTTAAAAGGAATAACACACAAACTGTGGTTAAAAACATCAGAAATAACAAACGCATTGTGTAACTGACTTTTATAGAGGTCTTTATAATATCGAACTTGACGTTTCGTATCCTTCAACTGACATCTTCAGAATTGACTGTTAAGATGAGTAGTAACTACACTTAAATAAAAAAAAGAACAAAGTAAGATAAGTAACTAATTAACGTGAAGGAAAAAACAAATAAATGTGACAGATGAGAAAATTTCCATGAAATTATGCAAACTAGTTTAAGTAAAGCTTTTCGCTGCTTATATTTTCATTTAGGGCTGGTTTTTAAAATCCCTAATAAGGTTATTTTCCAAAACACGCAAAACACACATCGCATCAAGTCTCTTTTCCCCGACAAAGACAGGCTCAACCGTTCTCAAATGTCCAATGTTGTATATAAAGCTAGTTGTTGGGACTGTCTGGATTTCTACATTAGCAAAACCACACGGAGATTGCATGACCGAAAAACTGAACACTTCAAGGCAATCACCAGTATTAACGGTCATTTATCAGCTATTGCAGAACTCGTCACTTCAACTGTTTACAATTTGAAATGGCACCATTTTGACATTTTAGCAAGAGGAAAATCAAATACCGATTGTAAAATCAAGGAGGCTCTTCTTATTAGGGATTTAAAACGAGCCCTAAATGAAAATATAAGCAGCGAAAAGCTTTACTTATACTAGTTTGCATAATTTCATGCAAATTTTCTCATCTGTCACATTTACTTCTTTTTTCCTTCACGGTAATTAGTTACTTAACTTACTTTGTTTTTTTGATTCAAATTTAATTACTACTCATGTTAACGGTCAATTCTGAAGATGTCAGTTGAAGCATACGAAACGTCAAGTTCGATATTATAAAAGTCAGTTATGCAATGCGTTTGTTATCTCTGATGTTTTTAACAGCAGTTTGTGTGTTATTCCTTTTAAGTTTATTCAAGAGCCATAATGACCATTATGGCTCTTAGTTCATAATTTTACTTTAAAAAATAATGTAAAAAGTAAGGGAGAATGCCCGCAGGGTATAGAGGAGTTAAGTTGATTTTCTTTATGGGTACCTGCAGTTGCCTGCAAACCTCGCTTTAGGTATGTGACTTCCCAAAGGGTAGGATTTTTGCGCCGTTTTGGTTTGAGAACGGGTACACGTAGGGGTCAAACCACCCCCCAAATGTTTCATGAAACCTAATTTTGGTTATTTTCAACATTATTATACTAAACTTGTGGTGTTTGCTCAAATTGATGTCCACTTCAATTTAAGCTGAAGAATTTCTCAAAATGGCACATAGCGGCGTCACAAAGGATGACGTCACAAAAACCAGTAAAGTGAAATTTTTAATTATTATTTAAAGTGTCTCATCTAAAAGTGGGTTAGCCTGGATTTCTTCCGATTACTTCGAGTCGTTATTACTCCCTCAGTAACGTCTGAATCGACCGGCCGTTAATGCCGCCAGTGACGTCAATACTCCTTTGCTTTAATTCATGCAAAAAGTTAAACACGATTTTTAAGTGGCAAACCAAGAAATATCGTTCGGAAATGTCTACATGATACAGAACGGTTGACAGAATTGCTTTCATGCGTTGCGTGAATTTCTCGAACGTTCTCTACGTTTTTGGATTATTCATGAATAATTAATTAGGGCATGTTTATATTTCAGCATGAGTCAGCAGATTTGCATCATTTACTGAAATTATAAAATATGTCGAAAAGCTACTACTATTCGCTTGTTTAGTATTTTCTAGAACTTTATGTCAAACGGTATACAAGTTCCAAGCGAGTTCTTTTGACGCTCAGGCCACTTAAGGCATGACTATCTAATTAAATATGCATATTTTGATAAAATGCTATTTAGAACGGTTTGCTTCTATCAATTATCCTAAAAATTGAACTGCAAATGTAGCTTACTGGCCTTAGCAATATGAGTGCATTTTAGAGGCCCAAATTTTCGTCACGTGACTGACAATTGAGAATTAACGGTCAAAATATTTACTCTAAAATGAGGAAAGGATAATGAGAGAGTAACAAGTTCTTTTAGGTGCATTTCGGAGACTACTAAAAAAGGTTCTTAAAAATTCGCCTTGTATGTGTGTTTACGTTATATAGGAATTGGTAAATAAAAGAAAAAGGGTCACGTGACTTATTAATGAGTTAAATAGCTAATAGGATAAATAACATTTTCAATCGTACAAGGCGAATTTACGATTTACACTGTTTATGTAACATTTTGGTAGTGAAAATTCCTAAGATTAAGATTTGCAACCCTTCCCCGTTTAGGAAAACGTGTTTTGTGACGTCATTGACGAAGCAATATCACGTGTTTTTTTCTTTGAGCCACATGTGTATCTCACTTTTCAGGATTACTGATGAAAGCAAACCGTTCTAAATAGCATTTAACAAAATATGCATATTTAATTAGATAGTCATGCCTTAAGTGGCCTGAGCGAACAAAGTTTTTTCCCACGAGTCTGGAGTGCTGTGTTTAGTCAAGTGTGACGAAGGTCGATTTTCAAGTTCTGTGTTCACATTTGGCGGAAGCCGTAAGATATCTGAAGTTCAAGTGAGTGGTTTGTTATTATTGGCAGAGGCTGTTTAGTTTTACTTAAGTTCAAGTCAAGTTTGTGTGCTGTGTTTGCTGGATGCTGTGTTTTAAAGCTCTGTAAAGACTATGTTCCTTTGGTATTAAACTTCCTTGTGTTAATCCGACATCCCTGCGTGCTGATCGTCAGTGAATAATTATCCTCAAAACATTCGAACTCGTAACATTGAGTTTTAGCCAATTCCATGCTCAACGTAATTGACTCCTTACCCATGTCTTACATATCTTTAATCAGTACATGAGACTGCTATGCTGTTTTTGAAGCCAGATGTAAGAAAAATAGAGAATTCTTTTTTCGCTGTTTGTTTTGTTAGACTTGTTATTCACCGCTAGTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCCCCCCCCCCCCCCCCGACGTCGGTTTTTAGGGCAACACAGCGTGGCAATGTTGGAACAATGTTGTAACCATTCGCAACAATGTTGCAAAATCGTCGTTGCGAGTCGTCTCGTGTAACATCACCTTAAAGATCTTGCATTGAGCATTGAAACTACCAACTGAGCGAGGCCACGAGGTTATATATTTAAACGTTAAGACGTCAGAGAATTGATTGATGTGTTCAATTTTTCCTCTGGACTCTAAGGTTAGCCATTTTCCTGGCTGCCAACAGAGCTGCCAACTTGTTGGCGTGGATTTGAACAATGGGGAAAAGAGAACAAAAAAATTAGTCTGTAGTACAGTTTGAACTGAATCTGACTCAGCTTTTTCTTGCTTGTGTCTTCGAACTCGTTTCTAGATAAAGCACTGCTTTTCAAAGCACGAAGAAAAAATAACTTCTTTCCAGAGGGAGTATATGGAGTAATGACGTACTGCATAATCGCCATTGACAAAACGCTGGCTATCATGTTTTATGTTCCTCATGATCAAACTTTTGACAGAAATTGGTGGAATGTTAAGCTTTACAGTGGATATAAAAGGAGCAACTCTCAAATGCTTCACGAAATGTATGAAGATCATAGCGCTATCGAGGGTGATGCCTACTGGCACTATAAAGACCTGGGCCTGGGATCAGAGCTTAGAGCAACTGGTGCCATGGCTTCCTCTGGTGCAGCTCATCTTATGATTGAAATAAGCAAGCAGCACCGAAAAAGACTTTAA
>gene-Peve_00000122
TTACTTATGCATGACGCGTACTGTCCTATTAAACTGTCCTATTAAGGCTGAAATCAGGGCAGTTGAGAACCAATCAGATTTGAGAATTTTGTTATAGTTATGATTACAGACCGAATTGGACTCCACTCAGTCCTGTTACCATTACATACTACTGAGCATATACACACGGGAAAACGCGATACTTATCTTAAACGAAAATTCACGGGTAGCCTGCGAGGCATGTTAATACTATCGATGCATTGGTACCGGAGAAAGAGAAATTCAAGTTAGTTCCAAGGGAAAATATTTCACCCTTCAATGTCAGCGTCTTTGGTTGTCGCCAGCACTACCACTATGGTATAAAGAGAGATCGTATAAAAAAAATAAACTTTGCTTGAACATATTTATCTCGACAGGAACGAAAAGAGCAAAGGACCAAGCGTCAAGAACCAGGTCAGTAGTTCATGTATGACCCCATGAAATGTCCTGAGACACCAGTGCCGTGTCGGGTCTCTAGGCACAGAAGCAAGTGTCCAAGAACCAACACGTAATATACCTTTCATTTCCTTCTTTATTGTGTAACATAGGTCAGGTGTTCATACGTTAATTAATTATTTATTTATTTATTGAGAAGCAATAACAGACCCCATGCAAAGGTCTGCTCCCAAAATACAAAAGAGATCCTCAAAATTTTTTTCGAAATTGTATGGCTTAAGACATCTCGAAGAGTCTTTCGTGTTGGAGCCCAGTATGGACCCAGTGGAGGCTGCGAGTTTATATATTATAAATTTGCCGCAAGTGGAAGGTATCTTGAACATATGCCATGCATTTGTAGGGACCGCGGAGCAGGGTTTGGAGTTGTGGGGGGAGGGGGCAGTTAATCTGAACCCGTAAGGGTGAGCCTACTAGGGGATACAGGGGCATGTTTCCCCTGTAAAATTTGATTTTAGGAGTCCTGAAACGGCTAGAAAAGCACCTAAAACTATTATACTTAAATAAAAGTAACGCAAGGCCAAATTTTTAACGATCATATCGCTGCTAAAGTCTGTGAAAGTTGTAACCGAGCTGGTGCTCCTTCAACTGGAAAAGGCAAGATAATTAAAAGGTATAATCGTACCAGTCTCACAATATTTAGGCAGTAAAATAAATCCAACTCCATCAATGACTTGTGCCTTGTTCGAAGTTGTTGCGGATTGGCCGCTTCATCTTCTGACAACTGAGTATCTGTCATTATTGGCTCAAACCTACCACACCACGCTGTTGAATATTTTTAAAAGACGTTTTCTGAACGTTCTTCCCTTTTTATCCCTACCAAAGCCTCTCGTCAGGTTTCGATCAGGTTCCTCATTGCGTGTTTCCAACTTGAAGTTTAAAACAGCATTGATAGAAGAAAGAAGATTCAATAACAAAACCTTCGTAACGTCCCAGTGTAACAAAACCAAACATTCAGGCCTCCTGTTGTTCAATAGGTGGTTAGCGCTACTCTCTCCTCCGCAGAGGCGTCTTTGTGTCGTAGGGAGGCTGGGGAGAAAGAAATAGAAAGCGCGCGGGGCACGGTGGGAAGGGGAGAGAGAGAAAAGAGGCTCCCGTCTTTTCCCTCTTCCCATCTCCCCCCCCCCTCCCCCCCTCGCACTTTTTATTTTTCGATTATTGCTATTTTTATTGTGATACCCAGCAAGAGCCTCTGCGGAGGAGAGAGTTAGCGCTAGCCGACGGATAAATCACTATCCAGTGGATAAGTATTAGGGGTGAATCATTTGAGCTATCCGCTAGGTAAAGATTTATCCGAGCGCTATCCACCTTTTGAACTACTGAAGCTGGCGGTAAAACATTGATCCAAAGGCAAATTAATTCTGGAAAGAAACAGAAGTTGTTAACTCCATTATTTTACCGTCAAAAATCCAAGCGTCCTTTCAGCAACAATAAGCCTTATCACGGTTGTCGACGACATCTTTTGAAAAGTGGGCAACGGCGTGAGAAATTACAGTGTTTGTATGAGAATCTAATATCGAATAATTTCCCTAAAACGGCGGTTCTGAAAAAGATATGAAGTGTAAACTAAAGCTTTGACTCCTAACGATTCTACTGAAAAAATGTAAGGGCGTAACATGCAGTTACATTCGCTAGAACCACTTTATAAAATTTTCTATACATTTGTCTGTAAAAATTTCTCTTCCGACTAAAATTTTCCGGGAAAAATTGCAGACACCGTGAATATATGGAAAACCTAAAGCAAGCGTTCTGGAGAAATTGAAGCACAGTAACAGCCCCCAAAATTTGTTAGTAAAATCCTTTCCTGTGTAGCTTTATTTAATTCATATATCTTTTTTCAGAACACCCGTTTTACGGAAATTATTCGACATTAGATTCTTCTACAATCACTAATTTCTCACGCGGTTGCCTACTCGTCAAAATGGCGTCGAAAACCGTGATGAGGCCTGGAAGGAACAAAAAGAAAAAACAATAGTATTGCAAAGTTTACAAACCAGAGACCAGATTCCGGACAGTTATGTGTTATCACAGCATGGAATTTTTGTCACTGCAGGCGCAGACGTCCCTCCTGGCGAGACGTCCCTTGCGGCGAGGAGCGAGGAGGGAAGGCTGTATTCGCAGGCTAGCGATTGTGGTGATCGTATAGAAAAAATGCACACCCCTAAACGTTTTTGTTTGGCTTTTTTCACACATAGCTGACATAATCGCAGCGGCTGCACTAACTGTTGGCGTACTTCAAGGGATCTTGGATGGCATCATAGGTGTAGATCGCAAGATAGCCATCGGCATCAAGAATAGAAGCGGATATAGCTGGGGAGCAATTGATATTTACTTGCAACAAGGAGTTACAGATAGGGTCATTCCCCGTAGAGTCGATAATGGTAACTATGAATGTCACGAATTTAGCATGAAAGCGCTTATCGGGGACACTGTTATAATGTCTCCTTTAGGAGACGGGGTGGCCATTTCTACGTAGTCTTCGCCCAATTTGATTAATCACTTTTTGTGCTGGAAGAGAAACAAGTCAGTACGTGCCCCTCCTTCAAATTTTTTTCCATTACTTTAAGTTTTTGTTATGGTTTTAGCTTCTCCCTCTTCTTCTTACTTAGGATTTGAGCGTAACCCATTAAGGTAGCTCAAGCTCGAAATATAAATTTGACCAGAGCGCCGTTGTGTCGAAATATGAGGATTTGTGTGGGAAAACTGATATAGCTTTTTAAGGGGGAAATTCGGCTGTTTGTTTTAGGGGGGACATGGGGCTGATTTGAGAAGGAAAGAAATAAGGGAAGAAAAGGTATACACATAAATGACCAGTTAGTTTGGCATTTGTCTACTAATTATTCTCTTCATATTTTAATTATTAACCTCCCCAAATTCAATTTTCCATTATTTTAAACATTTTCCCCCAAATTTTATTCATCAAAACCATCCAAAAACAGTCGCACATTGAGCCAACGACAACGACCTTATTGAGCCTTTTATTATTCAATGAAGTCTTGTCTAGATTTGGAAGAAAAAGAGCAGAAATAACGAAAAACAATGCTGTTGAGTAGAATTCCCCCTTTACTAAAACTGTAAACCAGTATTGAACAAAAACTTAACCAGGTCTAGTTCTCTTTTTGTTTGCCATTTTGCCTGATGTGTGGTTTATTGGTGGCCTTTGGTCCACTCCGAGTTTTGGTTTATATACCAACCCTTTAAAATTAAAGACCCAAAGGTCAGGGGCACCCAACGAGAATATAGTTCAAAACCACTTAACATAGCATTTTTGAACGTAATTTAGTCTTTAAACTGTAGATATAAACATATTTTTATCCCCTAACAATTTTTCATCTGTTCGGATTTCCTAGGTGAAAATCTAGGGATCCGAAAATTATAGGGATCAAAACTTAACTTTTCGAAAATTTCAGTCAGAAAAAAGGCTCCCGAAAATTCTAGATGACCTTTTTAGGGTAAAAATCCGCTTAAAATGGGCAATTGTAGCATTTTTTACATGTTCGAAAATCCTAGGAGAGGCAGGCAAGCAAGAAATTTTATAACAAATGTTCCGAAACTTCTAGATCTCAAATCGTCTTCCGAACAGATATTTTCCGAAAATTGACGTTGGGTGCCCCTGAAAAGTCGATAAGAATTCACGCAGGACGAAAAGAAACAAAGAACTAAGCGAGGTAGAACCGAGGTAGGTTATTTCACAATGCAACACTGTTGCCCAAATGTTAACCATATCGGCTTTTCCCCTACAAAACTCCATATTTCGGCTCATATCTCGAGCTTGGGAAAAAACCTCTTCGACAAGTTATGATGTTGTTTTCATGATGTTGATGATAAGTTCATTCAATATCGTAAATGCTCTTGGTTTTTGGCCATCTCAGTTTTAAAAGGAATAACACACAAACTGTGGTTAAAAACATCAGAAATAACAAACGCATTGTGTAACTGACTTTTATAGAGGTCTTTATAATATCGAACTTGACGTTTCGTATCCTTCAACTGACATCTTCAGAATTGACTGTTAAGATGAGTAGTAACTACACTTAAATAAAAAAAAGAACAAAGTAAGATAAGTAACTAATTAACGTGAAGGAAAAAACAAATAAATGTGACAGATGAGAAAATTTCCATGAAATTATGCAAACTAGTTTAAGTAAAGCTTTTCGCTGCTTATATTTTCATTTAGGGCTGGTTTTTAAAATCCCTAATAAGGTTATTTTCCAAAACACGCAAAACACACATCGCATCAAGTCTCTTTTCCCCGACAAAGACAGGCTCAACCGTTCTCAAATGTCCAATGTTGTATATAAAGCTAGTTGTTGGGACTGTCTGGATTTCTACATTAGCAAAACCACACGGAGATTGCATGACCGAAAAACTGAACACTTCAAGGCAATCACCAGTATTAACGGTCATTTATCAGCTATTGCAGAACTCGTCACTTCAACTGTTTACAATTTGAAATGGCACCATTTTGACATTTTAGCAAGAGGAAAATCAAATACCGATTGTAAAATCAAGGAGGCTCTTCTTATTAGGGATTTAAAACGAGCCCTAAATGAAAATATAAGCAGCGAAAAGCTTTACTTATACTAGTTTGCATAATTTCATGCAAATTTTCTCATCTGTCACATTTACTTCTTTTTTCCTTCACGGTAATTAGTTACTTAACTTACTTTGTTTTTTTGATTCAAATTTAATTACTACTCATGTTAACGGTCAATTCTGAAGATGTCAGTTGAAGCATACGAAACGTCAAGTTCGATATTATAAAAGTCAGTTATGCAATGCGTTTGTTATCTCTGATGTTTTTAACAGCAGTTTGTGTGTTATTCCTTTTAAGTTTATTCAAGAGCCATAATGACCATTATGGCTCTTAGTTCATAATTTTACTTTAAAAAATAATGTAAAAAGTAAGGGAGAATGCCCGCAGGGTATAGAGGAGTTAAGTTGATTTTCTTTATGGGTACCTGCAGTTGCCTGCAAACCTCGCTTTAGGTATGTGACTTCCCAAAGGGTAGGATTTTTGCGCCGTTTTGGTTTGAGAACGGGTACACGTAGGGGTCAAACCACCCCCCAAATGTTTCATGAAACCTAATTTTGGTTATTTTCAACATTATTATACTAAACTTGTGGTGTTTGCTCAAATTGATGTCCACTTCAATTTAAGCTGAAGAATTTCTCAAAATGGCACATAGCGGCGTCACAAAGGATGACGTCACAAAAACCAGTAAAGTGAAATTTTTAATTATTATTTAAAGTGTCTCATCTAAAAGTGGGTTAGCCTGGATTTCTTCCGATTACTTCGAGTCGTTATTACTCCCTCAGTAACGTCTGAATCGACCGGCCGTTAATGCCGCCAGTGACGTCAATACTCCTTTGCTTTAATTCATGCAAAAAGTTAAACACGATTTTTAAGTGGCAAACCAAGAAATATCGTTCGGAAATGTCTACATGATACAGAACGGTTGACAGAATTGCTTTCATGCGTTGCGTGAATTTCTCGAACGTTCTCTACGTTTTTGGATTATTCATGAATAATTAATTAGGGCATGTTTATATTTCAGCATGAGTCAGCAGATTTGCATCATTTACTGAAATTATAAAATATGTCGAAAAGCTACTACTATTCGCTTGTTTAGTATTTTCTAGAACTTTATGTCAAACGGTATACAAGTTCCAAGCGAGTTCTTTTGACGCTCAGGCCACTTAAGGCATGACTATCTAATTAAATATGCATATTTTGATAAAATGCTATTTAGAACGGTTTGCTTCTATCAATTATCCTAAAAATTGAACTGCAAATGTAGCTTACTGGCCTTAGCAATATGAGTGCATTTTAGAGGCCCAAATTTTCGTCACGTGACTGACAATTGAGAATTAACGGTCAAAATATTTACTCTAAAATGAGGAAAGGATAATGAGAGAGTAACAAGTTCTTTTAGGTGCATTTCGGAGACTACTAAAAAAGGTTCTTAAAAATTCGCCTTGTATGTGTGTTTACGTTATATAGGAATTGGTAAATAAAAGAAAAAGGGTCACGTGACTTATTAATGAGTTAAATAGCTAATAGGATAAATAACATTTTCAATCGTACAAGGCGAATTTACGATTTACACTGTTTATGTAACATTTTGGTAGTGAAAATTCCTAAGATTAAGATTTGCAACCCTTCCCCGTTTAGGAAAACGTGTTTTGTGACGTCATTGACGAAGCAATATCACGTGTTTTTTTCTTTGAGCCACATGTGTATCTCACTTTTCAGGATTACTGATGAAAGCAAACCGTTCTAAATAGCATTTAACAAAATATGCATATTTAATTAGATAGTCATGCCTTAAGTGGCCTGAGCGAACAAAGTTTTTTCCCACGAGTCTGGAGTGCTGTGTTTAGTCAAGTGTGACGAAGGTCGATTTTCAAGTTCTGTGTTCACATTTGGCGGAAGCCGTAAGATATCTGAAGTTCAAGTGAGTGGTTTGTTATTATTGGCAGAGGCTGTTTAGTTTTACTTAAGTTCAAGTCAAGTTTGTGTGCTGTGTTTGCTGGATGCTGTGTTTTAAAGCTCTGTAAAGACTATGTTCCTTTGGTATTAAACTTCCTTGTGTTAATCCGACATCCCTGCGTGCTGATCGTCAGTGAATAATTATCCTCAAAACATTCGAACTCGTAACATTGAGTTTTAGCCAATTCCATGCTCAACGTAATTGACTCCTTACCCATGTCTTACATATCTTTAATCAGTACATGAGACTGCTATGCTGTTTTTGAAGCCAGATGTAAGAAAAATAGAGAATTCTTTTTTCGCTGTTTGTTTTGTTAGACTTGTTATTCACCGCTAGTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCCCCCCCCCCCCCCCCGACGTCGGTTTTTAGGGCAACACAGCGTGGCAATGTTGGAACAATGTTGTAACCATTCGCAACAATGTTGCAAAATCGTCGTTGCGAGTCGTCTCGTGTAACATCACCTTAAAGATCTTGCATTGAGCATTGAAACTACCAACTGAGCGAGGCCACGAGGTTATATATTTAAACGTTAAGACGTCAGAGAATTGATTGATGTGTTCAATTTTTCCTCTGGACTCTAAGGTTAGCCATTTTCCTGGCTGCCAACAGAGCTGCCAACTTGTTGGCGTGGATTTGAACAATGGGGAAAAGAGAACAAAAAAATTAGTCTGTAGTACAGTTTGAACTGAATCTGACTCAGCTTTTTCTTGCTTGTGTCTTCGAACTCGTTTCTAGATAAAGCACTGCTTTTCAAAGCACGAAGAAAAAATAACTTCTTTCCAGAGGGAGTATATGGAGTAATGACGTACTGCATAATCGCCATTGACAAAACGCTGGCTATCATGTTTTATGTTCCTCATGATCAAACTTTTGACAGAAATTGGTGGAATGTTAAGCTTTACAGTGGATATAAAAGGAGCAACTCTCAAATGCTTCACGAAATGTATGAAGATCATAGCGCTATCGAGGGTGATGCCTACTGGCACTATAAAGACCTGGGCCTGGGATCAGAGCTTAGAGCAACTGGTGCCATGGCTTCCTCTGGTGCAGCTCATCTTATGATTGAAATAAGCAAGCAGCACCGAAAAAGACTTTAAACATTAAGTCATGTATTCCCTTTATTACAATAACGTGTGTTTGTTTTTCTTTAATAAACTGAGATAATCTTAAGTCTAGAGGCCTGGCCAATTTGTATAAACTCCTCTAAAAATATCCTCAGAGTTGTCCTTAAATTTTAAATGCTAAGTTTTGCTTTATGTCAATGACTTTAGTGGGCCTGTTAATGGAAATAGCGAGAACGTTATTCCATGGATCCTACCCATTTGTGGCTGTACGGATCTGTCCTAAATGGTCGACAACGATTTAAAGCTAAAAAGGCCGGGATTTTCCGAGTCAGGGCCAATTAGTAAGTTCAGCATAGCATGAGCATGCCAGTTGCTATGAAAATGCGGTCTATATACTTCTCTATAAAAACCTTAACTTTTATTTATCCTTTCACCCTGCAATTGTTCATTAAATATTTCCTAAAATGTGTGCCAACACAAGGGTATTATAAATTAAAGGTTTCCAAAGAGTGGTATATATAATGCACCGATAATTTTCAAAACTTTGGGCGTCTACAACAATGAAAGGATTCCTGTGGGTGTCTCTACAAAGTTTTCGGCTTTTGGGCAACATCAGAAACTCATAAGCATATTGAACAGCAACTAACAACTTCTCATGACCTGTTTTAGGAAAAAAGAAACCCGGCCCTAGCTATCTTTAAATCGTAGCACGTAAATCGTTTAGGGCAGATAAGTATAGCACAGACGAACAACTGACTCTCAAGTTGCAAATATTACATATTTGGCTTATTACCAGCAGTTTCTATTATTTTTCCATAAGTCAGTAAAGCCGTTTTGAAAAAATGTCTAATGTTTAATCTGACAGAACGAGAAGTTGATTAGTTACTTTGAATTTTATCCAAACTGCTCTAGCCTTTTTTAGATTTTTCGTTTGCGTGTGTGTGTGTGTGTGTGTGTGTCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTTTTGTTTGTCACGATATGGAGCCTATAGCTCCGTATACCACTGTTAGGCAAAAAAAAAGGGAAGGACGGAAGGACCAGTTGAAAACCCTCTATTTTCGTACACTTTTCAAAATAAAAACCTCGAGTTTTTATCCGAGACGTTTTAGAAAGGACTAAATGTTCTTACATTATTTTGGCGATTTTAACTTATTTGTTGCATTGTTAACCTTGATACAAAATAGTGCCATCAGTCGATTGGGATTATTTGGACTATACTTAAGTTTAGACCTAACATCGTCAGTTACTTTCTATCTACGGTAACTTCATTATAACGTTGTATGCTGCACCGCATTATCTTGGCCTATGTTCTTAATATCTTCTTAAGTTTTGTCCATTTTCAACGGCTTGAACGTTCTTTTGCTGTTCTAGTTTTCTCTTAAACGAAATGAAACTGGTCATTTCGTATCGTGCTTTCCACGTTTGTCTTTGAAATTAACGCAGTATCTTATTTTAACGTAGAGAATGTGTGCAAAAATCCGTTGAAAACTAATGGTTCTTATTTGACATTAACCGTCGTTAGTGTTGGAGTTCAGTGTGTGAGCATTAATTCAGCTGATCAGAATAAAAGCATACATTCATCCTTTTATGACTCCGTATGAGAATTTTTGTATTAAGAATACGAATTAAAACATAACTCCACTTTTACCATTTAATTCGATCTCACCTAGGGGAAGGGGGAAGGCATACACATAAGGGAAGTTTGGGTAGGGGTCGAGACAATCCCCGTTTAAAAAATCTGTTTAACAGAGAATAAGATTTAGCAAAAATAATAATAAAATCGTTGGTACCACAATGATTCAGACCGCGCACCACCAATGTTCACACCTTAAAGGCGCTAAATAACGCAATTGTATACCCTCTTTAAGACTCAAGACCCAGAAAAACAAATCCTGTTGGGCGGGACATACCCCGGTTTAGGCACCAGTCAGTTGCATTGTCTTCTAAGGGTAAAAACTCTTTAAATGTTCACACCCTCCATCGTTCATATAGAATCGTTTTCGCCATTCAAACTTCATTGCATTTGCATTTAGCAAGAGAATTTTAAACTTCACTTTTTGATAGTTTCGAGAATCGTGATATAAAAACTGGCCCGAAACGTGCTGAATATCTTTAACTAACTGACAACAAAATGAGAACGTGATAAAATTATTTCGTCAACATTTGTGAACTTTTGCCCTCTCACCTCTTCTGAGCATGTTCCACATGTTACCACAGCTCAGGAGTGAGAATCATTGTAGCGTAACAAAAATCATTATAAGCTGGGTTTTTGAGGCGATTTTAACGGTCGTCACTTAGGAAAATTGTAGAGAGAACAAAGAAAGAACTAAAACTCGCTGGAATAGCGCTGAAAGTGTCAAAAATGCTAAGAACAAAACTAGGAGATAAGGTAAGGAGTTCTAGAGTTTATTTTGGACATCTTAATAATAATCACTAAACATGCTAATATCCACATAATATTTCTGTTTTCTTACTCTTGAGCTTGTCACCCCTCTCTAACAGTTATCAGAGTGTAAAGTCTCATTATGATATAATTGCTTTATCGCCCATAAAGGTATGGAGAAGAAAGAAAATGATCAAAAGTCTTCCTTGTTAATCAGTTTCTGGTTATCATAACTGTAGCAGAAGTACGGAAAACAGCTAGTGAGGAAAATACGACACATTTCCAATGATATGGCAACAGAACGTTAATATATTTTTAGGCTTCCTTGCGAATATCCAAATGCAGTAATTATGTGTCAGTTAGTAAGATGCCAAAAGGACGTGCGATTTCACTATGTTGACTATATTGCCGAGGGATTCTTAATCCGATCATACGGATTGGGGGATAAATCCTTTAGCAGTTTGACACTGTCCGGAAAAAACCTGTTCAATTCTTAACACGACTCCTGAACACAAGATTTTACTGCACCGAACAACTCCTTCATTGTTATGTGTCAGAGCAGCCAATAAAGACGCGTGTTGCATGAAGCTGGCAAAACTCCAAATGTAAACAACATCCGGGATGTCAACTTGTGCTTGTATATTTACTGGAGCATCGAACGTCCCACCCAATTTTACGCAAGAGCCGTGTTTATTACTGATCTTGCACCTCTGTTCCACATCAGTTTCGACTCACCAGTTCCTTCGCCTAAATCATGGCAACCTATATTCCATTAGTCACACCACTGGGCGAATTGCCGGAATTTCCCGAGGAGGAAAAGATTTACGACCTAAAAATGGAAGGTGGAGGAGTCCCACCAGAAGCGAATGAAGTGTTCGTGTACGTATTTGTCACAACACACGGGGAAGGATCTTTTCAGAGGGGATACTACGAAATTTCTACAAACATGCTGGGAGGACCAGATCTCAAGCAGTACATGAATGTGGCGACAGGGCAAGGCTTGTTGGCCGTTAACTCTGCCAATATGTGGTTTCCTGTTGGAGCTGGCCAGTTGAAGGTAAAGTTGGTCCACCCAGGTGAGGCTAAGAAAAGCATTGCAGGGAAGACCGACGAAAAAGATTGGAGCGGAGTGTTCATCATTGGATACCGATAGAGTGTTTGCTAGTTGGAGACCCGATCCGCGCACTTTCAATATTAACCAAGGAGCTGTTAAAACAATATTGTAGTTAACAGAGCAGAACTAATTGCATAAGACAAGGAACAACAACTAAAACGTAGAACTGACGCTCAGTAAGTTTACAGTAGAGGTGGCCAAGAATCAGTGTTTCATAACACGATTCTTTCTGAACAACAAGATATTCACCAACATGTACGCCGTAACACATTTTTAAAATAAAACTTGCAATAGAATTTACAATTTCTTTCCTGTTGTTTGTTTATTCATTTCAGGTTCAGAGACATGCACTCCCGCACACACAGCATTTAAAAGAAACTTTAACCCTGCGTCTCAACCCACATATTCTTCACACTGTTCACAATTTACTTCTTTTGGCTCTTTTAAGGAGAGCTCATCTAAATATTGGAACATTTTACTTTTCTTGATTATTTTCTCTACTCACACTACCTTCAATGTTTGAATTAGAATTCGTACGATAGAGAGAGGTTAGATGCTGATCACTGATAGGGTTGAAAGGTATAAGGGTACCTCATTTTTAAGAGACCGTATCCATTTTGCGGTAAGATGTGTCGACGGATATTGGCATGGCTTTGCATCGTACAAAAAAGGTAATTAGTTTAGGATGACCAGGCCGACCCCCTAATCTATAAAAACACTTCAACCATTTAACGCCAGATGCACTTGCAACTTATAGTACTGAAATTCATGAACTTTTGCCTGTTATGTTAATGGGGAAACGCATTTCTGACCGTTAATGAAGACTTGGTCACGAAAACTATTTGCAGGTGGATCATCGTTTAAAGAAGGCTAAGTTCAACTGATATATAAAAAATATATTGATACATACAATATATAATTCGTGTTAAGTTGACATTTGTTTTAATCGGCGTCGCTGTCGTCGAAAAGCTGTAAAGGCAGCGTGCAAACGGACGCAAACGGACGCAACAACTCCCAACATTAGAGGTAAAGACAAATTTTGAAACATTTAGCTCAAAAAAGGTCGATTAGAAAAGGAAGTTTTAACCCCTTTACTCTATTTACTTTCGTGAGCACTTAAGTGGACAATTAACTGATGTGCAAATACAAAAAAGTAGCGGGTTGATTTTTACACGGCAACTGTCTACTACATTAACTTAGCGTCATTATTTCACGCACGCTCTTAAAACGGGGGGTAAGAAAAATGGGAAAGAGATCCATTTGCTATTGCTCGAGAGAAAAAAAAACCGCTCGTTTTTCCCCACACGGGGTAACTTTGCTCTAATTTGTTAAACAAATTCAAAGCAGTTAATAATTTTTATTTTTTGTTTGTTTTTTTTCGACAATATATGAATTAATGCAAACTTTTACTAGCTGTGTCGCTCGCTCAAATAGATCGATTAATGGAGGAACTGACTTTTAAAGTCAATATCGTTTTCCTTTACTGCATTAAAATTGACACGGGATTTGGTCCTCCAGAGAGGCTTCTTAGTACACTAGCAGGCTGAGACCAGGGTAAAAACAAACCGCAGGGAAACAATGGAAAAGCCCAGTAAATAATTTAATTTATCACCTTTTTGTTTCCAATATTAGTGAGAGTAGACTGAGTGCCGGTGCTCCAGCCCCTCTAATAAAATCGGGTAATTTCTCTACAGTGAGAACCTCTGAGAAAGAGCACACCGGCGGATAAAACTAGTAGTAAAAAAAAAACAGATTATGGGTACTTACCCTGAAACGTCTTCTTACCGCATCCCAGCACAAACTGAATGATGCTGGCAAGCTCGGCCTCTCACACGCAGACACAAATACAGTGCCGTATACATACTAGTAATAACCAAAGGTTACGTAGTGCGGGCGACAACGATCGAAGGTAAAATCGCTTTTGCTCCAGCGCTGTGCTTTGCGTAAGTTTCACGTGACTGATAGTCAAAACATGCATAACCAGATTACGAGTGATAGAAGGTCCAAACGCGCGTGATCAAAGTGTGTTCTGTGCATTCTATACATTCATAGTGGAAGTCCAAACGAATGCTGGCTACATACATGCGACGCATGCGTAATAACAACCTGGAGATTAGGATTGCGCGCTCCTCTTGTTGCCCGCAATTAGAATTGTAAATAGGACATTTGCATGATTGCGTCATTTTACTACTATGACCAGGATCCTTCAGGGTTTTGCTTTCTTCTGCAAATTAGGGCTTTTGTTATTTAAACCTCACTGGGATTACCATATTTAAATATGAAAAGAAAAACGAAAAATTCTGGTCCTAGTAGTAAAATGACGCCATCGTGCAAATGTCCTATTGTAGACTGGTGTCTGCTTTAGAAAGCTCCCTCTTGACATTTTTCATATGAGAAATAAAATTGACTCGACGTCATTAGTATTAGAGATTGTAAGAGAATTGTTACTTTTTAATTGGCACATTTGTTTCTATAAACATGCCAATTAAAGGTCACTAGATCTCACTAGCTCACAGGTACTGGCAAAGTTTCATTCTTTCCCCAGTCTCCCCATTTGGTATCGTTTAATATCTAGTCAACAATAATACTGACATGAGAATTTTTATGCCACACTTTATTTTTCTTTTCGATGAAGTCAACGTGTCCATTAATCAAGTAATATTAGACTATTATCTACTTGTCTTTTACAGGTATTAGGTTTATTTACATATATGGCTGGTGGGAGTTTCATCAAAGAGACATTCAGAATTGTAACTTTTGAAAATATCGTCCTCATAATCTTATATCACAATCGCTGTGATTCACTGATTATATTTCGTATTTGCTTCTTTACCTTCTCAGCTTTAGTCCTTTCCGTCTTTTTAAGGTCACAAAAACATTCTCAAATTGATGACGATGAATAGAAATCAGGAAAACACCTTTTAGTGCATGATAAATATGCTAACTTAGACTCTTCCAGGAAAAGAAGTCTTAAAATTAGCTATATACTTTGATTCTTTTGGGAAAGTTTTTCTCTCGATGTATAACAATCAGAAAAATGTTTCATCACATTGACCACCTTGGTTGTCCCATTTAAATTTTTGCAAAGGAACTTAAATCTTTTTTAGAAATGAAACTTTACATATAAAGTTAACCAATGACAAGAGACTATTTACTGCTTCAGTATGTGAATTTCGATGGTGGCTTGACCGGAGTTAGACATGGCATCCCTCAATCGGAGTCCCGATCCCAAATTTCTATCGTTCCAGTAGTTATCTCCTTTGAAGGGATTATCGTAATACATCCGGTACCAAAGATTGTAGTTGGCGCGTCTTTTACCACTGTAAAGCCAAACATCAAACCAGTTGCTGTACCAGTTGTAGTCAAATGGAACGGAATACATAACAGCAAGTGTTTTATCGATGTGTGGGATGTAGTATGTTAATACTCCTACTGCGCCTCTCGCAACGGGCCCCGCAGTTTTTCGTGCGCCGTAAAGCAAAGCTGTGCCTAGAAATAAGCAAACAAATTAGCAATTAAACAAAAATCATAAACATGATCATTCGTCACAATGATACATGGCAAACGAGCATCACAGAATAGAGGCATGTACCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTGCTACTACTACTACTACTACTACCACCAGAATCACTCAGGGTTTTGCTTTCTTACAAACGAGGGCTTTACTTTTCAAACCTCGCATAACCTGTGATCAGGCCCCCAAAGCAAAACGGGAAGAAGGACAGCCTTATTACAGTTTAAACTCTTCGCAGAGATTGAATAGATTCTATAGCAATAGATCAGACCTTTTTAGCTTGTATGTTTTGTTTTCCCATTTCAGACCAAGTGATGGTACCCCAGAGAACTGTTTCTTTCAATGTCGTCTTTTGCACGTGCACCCACATGTCAGGATGTATGCATAATTATATAAAAGACAAATTCCCCGAAGAACGTTGGGAAAACAAAACAAACAAGCTAATGACACCATCCTGCATACGGGCCTCAGTGTCATTACTAAAACAAACGGCTAGCCTGTGCAGCCTTACGTTTCAGCTTCTTTTTGTTTCTCCCTTTTGTTTTCAAAGGTTATGGTACGCAACTTCTAAACAACCGATGCAATTACCTTTTCGCTTGTTTTTCTCCTCGCCTTCTTTACGAGTAGAGATCAAGGACGAGCCCTGTCGCCCCTGTATCTGTGAAGGAAAGTATGTCCATTAAGGTCACTCCACTGAATACAAACTGGAATGAGCATTCTGTTTTGTCACCAATTTTTCCTCTGTGTATTTCAGTACATTTGAGGCACAGAAATGAAATCGGAAGCCAGCTAAAAATTAATTATCGTTGCGAGCCTTAGCGAAATCACCAAGAATTGCGTGATGGCTGTGTATTCATTCAGTTTACACTTCGCCAAACTGAACATTGATTATTGAGGGACACACATCGCAAAGAACTCAATAGATTTACAGCATAAAATTATAAAAATGTTTATTATACATTCAAACGTTAAGGTGTAAAACTACCTAGTATCTACTCGCAACTGGAGGGGGGGGAGAGGTTGGTCGGTTTTATATTCGCTCGGAGATTAGGGTTCATTTTGTTTCACTGAAACAACCAAGATTCGATTAGAGCATTGAGTTGCATTTCCGTAGATATCACGCCACCCCCAAGAGAAATTTTCATGTTTGGCTTTAATACAAGTCACCAATTGACTATCTTATTTTAAAGAGAACTATTTTACCTTAGTGTGTTTAGAGGACAATCCAGGACTCCTTTGGACATGATTGTCTTGCTTGAAGGGAGACCCAAAGATTGACTGAGTTATCAGCAGAACGAAAAACATAAGGGTAAACAGCCTAACTCTACTAACTATGGCCTAACGATGCCCATTTTCAATTCTATAATAGGAGAATAAGAATGAAGGAAAGAACAAATGCAACATTGTTGTCCCGGTACAATATCATTTTAAGAGGGTTTGTTTGTTTGTTCGTCTGTAAATTGATGAGATGAACGATGTTATGACCTGGGAATGTCATAACTCATACGACAAGTTCAGGTAGCGCTCGTGGTAGAACTGATTGATGATAAATCACGCGACAGTAGCTGCATTTGAGACGAGAGACTGTGTCATAAATTTGCTGTCGTTCTTGTTTTTAACTTTAACTAATTAACTTAACGTAATTAATGTAATAAACTACCAATGTTGCCAATAGCTAGCTTTAAAAATGCTGGCACTGAGATGATATTAGAGAGATTTACTCTTACATGTTTTATTGCAAACGGCAAACGTGAATTTGTACCACGTGACCAAGTTTTACTCTTACTTGACTTTTACAAAATAAGAGTTGTTTGGGACAGTTTTTATCTGCTCATTTATTATTTTGAATCTACAACTGCCGTAAACGTGACTCTGAAGTTTCGCTACCCCTTTCGACGCCTGCTACGCAGGTTAAAGAAGTACAGTGGAACCCCGCCTTACGGCCACCTCGGTAACATGGTCACCTCGTTATTACGGCCACTTTTTTTTGGCCGCCCGGCTAAACGACCATACATTTTCTTGTAAAAAAACAAACCCTCGTTAATACGGTCACCCGTTAATACGGCCAAATTTTTTTGGCTCATTGGTGACCGTATTAACGGGGTTCTACTGAAAACAAACTTTTCTACTCCGTTCTCTGGAACGGAAAGGGAAACCGATCATCAAGCGAAATGTTATCTTCAATGACTATCCCAACGGGGAGCTTAAAATGATAGAAACTGACTCCTTTAGCAAACCACTGAAAGCTACTTGGATAAATAAGTATTTGGATGAGAAAAACAAGAGTAAATGGAAATTTTTTTTTTTGGCCTAGAATTGCTGAGAATTGGAATCTTTCGGTGGAGAAACTGTCCTCACAGGTAATCTTAACATGACGGACACAAAGACTTTGCTCAAAACAAAAGATAAATTCATCAATGAAGATCTGACAATTTGGGCAGAAGTAAACTTTGAAGTGCAAATCAAATCAGACAATCATTTTCTTAACCAAAGCTTCGCTTATGGCACAACTCGTTGATAAGAATTGACAACTGCCCTTTGTTTTTCCCTGAGTGGCATTGTAAAGGTATCACTAAGGTAAGACACTTGAGACAGGATGACTCAAATAATTTTTTAACTCTCCTTGAATTACAGACTAGTATTGGACTAAAAATCTGCCCTCTTAAATATCGTGGATTGTTGACTGCAAGCTCTCTGGAACGAACATACAGGTAACTTTTCTGCGGACGGCTCTGAATGTCCAATTTTTTTGGAAAGGGTGTCAAAAACACAAAAAGCAAGTCAAATTGTGTACGCTAAACTTATATGAAGAACTGAAAAGTATACTCCCTACACACACTCAGCAGAAATGGATAAAGGAATGCAATATCGAAGGCAATAAACAATAAATGAGGTACGAAGCCCGATCAGCTCCAAAATGTCAGTGGCACCCAACGTCAATTTTCGGAAAATATCTGTTCGGAAGACGATTTGAGAATTTTCGGAACGTTTGTTGTAAAATTTACGGTAAAATTTCTTGCTTGCCTGCCTCTCCTAAGATTTTCGAACATCTAAAAAATGGTATAATTACCGATTTTTAACGGATTTTTACTCAAAAAGGTCTCCTAGAATTTTTGGGAGCATTTTTTCTGGCTGAAATTTTCGAAAAGGTAAGTTTTGATCTTATAATTTTCGGATCACTAGACTTTCAGCTAGGAAATCCGAACAGATGAAAAATTTTTAGGGGATAAAAATATGCCTATATCTACCTTTTAAATACTAAAATACGTTCAACAATGGTATTGTTGGGTGCCCCTGAAATGTACTAAAAGCACAAGTCTGGTAGAATTTCAATTCAGACTTCTACAAAGACGAATATTGACCAACAACTTTTTACCAAAATTGGAATAAACGATGATCCAAACTGCTCCTTTTGTAATGAAGAGCTAGAAAAACTAACACATCTTTTTTGGTCTTGTTTCAAAGTAACTACCTTTTGGAATTCCTTAATCCAACGACTTACTTTATCCCAAATTATCCCACAAAATAAATATTTCTACGAGCCTTGGATTGACACCAGACGCCCCAGTCTAAAAACCATAATAAATAAATTTATGTCTATTATTAACAAGAAATTATATTTGGATATTTTTGTAAAAATAAGAAAGCCTCGCCTAATATACAAGACTTTCTTCAATTCCTTAAATCAAACTATCCACTGGCGAGGAGCTAAACTGAACCTTGTGATGGCCCCAGTGAAAAGTGGGAGCTATTGCAGACTTTAATTTTGAAAGATCTTAGTTTGTTGATGTTGCTCTTGTTTTTGTTTTATTTATTTTTTCTCTTTGCTCTCGTCGGTCCCCTAGATAAATTCGGTTCTATCCTTCCTTCACTGCCTTCTTAAACAAGTTTAACTGCCTTGATAATTTATCTGCATTGCATAGTATCCCAGTATTGTTAGTGTTAAATTGTAAGCTATAATTAGCTAGTTATTGTAAGTGTCAACTTGTAAAGTTATTGTTACTCTCACAACTGGTAGTGTCAACTTATTTGGTCCTGTTTACAAGGAGGGAAGGTAACCCTGGTGCTAGGGTTGTCCTATCACTCTCATATTTCCTCTTTTTTCACACGACGTGTTTACAAGGCAGGTAGGGTTACCCTAGCGCTAGGGTAACCTTACCTGAGTACAGTATTATCCATTTTCTGAGCTTAATTATAGACATCTGAAAAATAAAAAGTTATGTTCTGTCTACCATGATAATATTTCCCTTTTTCCCCTGAATTTAAGGTGTTTTCCATAGAGAACTTCAAGATTATTTCAAATTTTGGACCTTTACAAAGAATGTCACGCATGACGAAACACGTCAGCAAAGCTTGTAAAGTTGACCCGGGGCCCGGTTCCTGAAAGACCGATTAGTGCCAATCCAGGATTAAAATTTTGCTCCACTTTTTGTATTTACCTTCCTATGCATTGTCTAGAGTAGCATTTTGTGTTATAATTTCTTTTTTTTTTCGAAGTAAAGGCACAGCTCGAGTGACTTGTTCTTAGACAAGAAAGCGTTGCTTAAAGTTTGGCTTTATCCTAGGTTAAACTTAACCATCTTTCGAGGAACCGGGCCCCGGGTGATAGGGTAACCCTACCAGTGAAATTTGCTTGTAAACTAGAGCTAATTTTAACCCCCTTGCTAGGGTAACCCTAGCAAAAGGGTAACCCTCTTTCCTTGTAAACAGGCCCTTAGTTAGTTATTGTTAGTACAGTGTCAATTTGTAAACTAGTAGTTACCGTTTGCCAACTTGTAAGCACCGTTAAGGGTATTGTTTGCAATCTGCTGTTTGTTACTATAAAAAAAGTTTTGCAAATAATGTCAATAACTATTAAGTCTACGACAGATTTGTTCCTAACTTTACTAAATAATTCTCAGAGTTATCTAATTTTGTGTGCAAGTTTGAAGTCAATCGAGACTATACAACGCGCTGCACAAAATGCTGGTCAAATTTGTCCTAAAATGCCATGTAAACTTGTGAAAGCCGGTTTTCGTGCATATAATTGAAGATTACCGACAATCTACTAATCCTGCTATAGACTGCAAGCAGTCTCCCCAGGGGGGGGTGGGGGGTACCGCCATATATGGGCTATATAGGTATGTACCGTTGTGAAGGGTATGGTTTTCAAGCAGTTTGCTCTAGGATAGCGTATATAAATCAGAACATTTGGGTCTAGAATAGGAAAGGGTATATAAATCAGAACATTTGGGTCTAGAATAGGGTATCATTTTTCAGGAAACTGATCAGTTGGTTGAAGATTTTATCTAGACAAGGGAAACAGCTACTCTAGGATAGGGGGGATTTGGGGAGTTTACTCTAGTATAGGGTAGCAAAATCTAGCTGAACTAGCTCTGGTGTAGGTTAAGGGTTCCAGGGTCCCAGCGGCACATCCCCAACCAGAAATTCCTAAACTACCCCCAGGACAGTCTCTTATTTTTCTCTGCAAAGTGACCCAATGCACGCGAAACCGAAGCACGCGAGCGGCGAGCGAAGCCGCGATAAACGAAAGCGTAAGCCCGAGACGTCGTGGTTTGCAATCGCGCTGGCTGAGATAAGAACTAGACAGATTTAAGGGAAAGGGCATTTATTTAGTGAGTCGTTTGTTTCTGTCTTTTACCTTATTATTCCTGCCCCGTACCCAGACGTCTCTCTCGTGCGCGCAAAGGAAGGCGGGCCGTCTGTGCCCTTCCCATTGTCCCTTGCGGCTCATCACCAGTCGCTCGCCTCTACCTTGCGAAAAACGAAGCGGCTGAGGAGGTTTTGAGGAGGAGGCTGTTATTGTTTGTTGTTTTGAAGACAAGGCAAGTACTAGTGCCAGTCCCTCCCGAGAACTGAATTTTCAAGATGGTGTCCCCTTGTTATTTTCTAGTTTTATCCCATGGTTTAGCACTTTTTGCCGTTTCAAGATGTTACAGTGGCAGTATTTCGAACTTTTTGATATTTCCTCTACCTGTTGACCATGCTGCCAGCGAAATAGGGAAGAAAAATCATGAAGAGGACTTAAACAAACAAGAAGAGATTGGCCTATTTTCAAGAAAAAGCCAGTTGGTACCTATGCATACAAATTTGAGTTTTCTAATTTCTTCCTGTTCCAACAAGCCAAAATGATTATTAGTGTTTTGGTAACATATATGATAGTAGACTGCAATTTGGCTGTCAAAGTTTTATTATCCTTTTAGTTGTGCGCTACAGATCAATTAAAAAACGACCGATTTTTGGTTCGCTCGCAGACGCTGAGGTTAGGTCAGGTAAAATTAAAAAGTTTTAGTCTTGCCCGTCGCTACCTAACAATGTTTATGGTTTTCGAAGATAGTCCTGTGTTGTTTTGTATCGCATACAAAAAATGAACTTCGCTCGTTTAATTTTAAGGAGTTTAAGAGCTTCAGAAAATGGTCCAAATTTGCATAAAAAATGGCTTCAGAGGGGGCCAAAGTGATGCTCGTATCTCTAATATCTTCTCTAATACAATAGCAGTGAAGAACTTGTGTTTATTATATTTGTCTATTACTCCACCTAACCTATCTATGATGTTAGTATGAGCTTATTCTGTTGACCTCCTTCCGTTGACATTTTTAACATTTAAATTGCCCGGTTTTCTCTGACAACCACTCTTAAGCAATCAATAATTGATCCTGTAGTCAGAGCTCCACCATCACTGACACTATGGTCACCATTTTGCAATTATCCATTCTCCTTCAACAAAGCAGACTTAATGACCACTCAAATGGCGACTTAACAGTATCAAAAAGCGCTTCCAACCAGTTGTGAGTAAAATTTCTGGATAAAGCGAAACCAGACATAAGAATTCTGAATTTGTTTTTCATATTCAATAATTCACAGTTCAAAGCATTTTTCATGTTTGAGTCCAACACAAAAGAAGTTGAATTTAATAGACCTTGTCACGGTTTTCGACGCCATCTTGACGAGTAGGCAAACGTGTGAGAAATAACAGTGATTGTATGAGAATCTAATGTCGGATAATTTCCTTAGAATGGTTGTTCTGAAAAAAATATGATTGTGGATAACACGTGACCATTTTTCCTGATACCGTACTTTGGGCTAATATGGAACGAGGTCTTGAATCCCAAGAAATTCTGCACATTTTTACCACTGAAACTGTGACATTTCCCGGAGGAATCTGACCGTTACGTTTTACGTGTAAAGATGGAAACCAGGTTCCTTGCTGGCGTATTTATTTCTTTAACAGAAGAGACTGTGAAATACCGCCGTCAGGTTAAAATCGTTCAAACGGTCCAGCGCGAAACCGTGGCCACATAGCGACGGGTATGTCAAAAAGCTGAAGAAAGTCAGTTAAAAACGTTTTCTAGCTTGCGCGTCGAGCATTTTCGCTTTCCTTTTTTAAGGCAAAAGCTCATTTAGGCATTGCGATCAGCGAATCAGGCCGCGTTTTCTGAGAGTAGTCATGTAGTCAGAATAGAGTTCATATTAACTCTATCCCGTCCCATGGGAATAATGGACTTTCCTTAATAGAGATTAGAGCCATTATCCACATTACTAGTTTTTCCTTGTTTGCCATTATCCACCGGTATTGCGATGAATAATGAAGTATCTTTAATGGAGATTAGATATTATATTACCTGAGAGTAGTCATGTAGTCAGATTGGAGTTCATGTTACCTCTATCCACATTATTTCTATGAATAATGGACTTTCCTTAATGGAGTTTAGAGCCAATATCCACATTACTAGTTTTTCCTTATTTTCTATCATTAAAAAGTGATATCACATGTTGTAAGTTTCCTGCTGGTTCAACGTCTTTTAACTTTAAAAGTTAGCAAGATATAAAAGAACCTCATTACCTTTCTCATAAAGGAAAAGGTCAAGTCCAAAAATGGTTGTTTTGAAACCTTGTATAACTTGCCGATGATCTAACCGTCCTCGAAATGACGGAAAGTCAAATAAAAAAATATTTAACAATTATTCAATGAGTGCGCGTTGGATATGATGAGGTTGTAAATAGCCAACGAGGCGCGTAGCGCCGAGTTGGCTATAACTGCCTCATATCCAACAAGCGGGAATGAATTAATAGTTTTATTAAACTTTTTTAAACTCCAAACGTCTGGTTATTACAACTGAATTTTAGTAAATTACTAAACGACCGGATATTAATGTAACTTCCGGTAAACCAAGGGAAAATCACATGACATGTGCGCCGCTTGCGAACTTAGTTGAATGAAAAATCTGAAAGGGACGGACGCTGTGCTTTAATTATTCTCGACAAGTACTGTTTTCGCTTTACCTCGCTGTGACTGTGGAATTTGTCGCTGGATAGGTCGACCTGATTGCTCTTCAATTTCCTTTTCTTCCTCCACTGAATCCGAGCTGTAGTGTGTAGTTCATGGTTGTTGCTGTTAAAAGGAAAGCCGCTCGTTGTAGGATTCACTTGAGGCCCGTTATGGACATGCGAAAGTGTTAAGAGATGTAAAAGCTCTTCAAACAAGCAAAAAATCTGCCAGTGGTTTTGAAAACTTACTCCTACAAGGACGTGCTAGCCATTCTGAAAATTTTTAAGGTAAACTTTGTTGTTGTTGTTGTTGTTGTTGTTAAATAATCAGCTCGACGTAAGGAAGAATTAAGCTTATAATTTCTTCGGAGTACCATTAAATAATTTCAAACCAAAGTTTGTAGCTTTTTTGTCGCTGCCGCTATGGCATTTTCTGTTTTTTTTTCTTTTATTTCGTCGGTAGATCTACTTTTTTCCGCCGCTTTGTCCGAGGGAAATTTTGTTGTTTTCCATATTAGGTCAAATCAAGGTACATGAGCAAACATAAAATGATAACCGAGATTGAGTGAACCAATCAGAATGCTAGATATGCATTATCATATTAATGTGTACCTGTGTGGGTGAGCGTGTAAACCCCCATCGCAACTGTTTTCCAGGGAATAAAATGACGGCTTAAAGTTATAAATGATTATGTTCCGCGATGAAAAACACTCGACTTACCAGAGTTAAGACTAATTTCACCAGCGCGCATTGGATCTCTTAGTGATCGTTTTCACTGAAACGCTCCTCACCCCTGGCCTGCGCATACACTTTTCACTATGAATAACTTTCATGCTAAAATTTTGCTAAACAAGCACCGTAGATTTAAGAACGTCTCTTTCTTTTGCTAAGAACGATCATAGCCTGTTGTCGGGAAAACCGTTGTTGAAATACGTCGGTTTTCCGCGACATTGTTTGGGACAAACCATATATGTATCACAAGGGCCATGTTGCTCGCGTGACTTTCAAGAGCTCAATCATAGGAGAAAACAAACAACACTTAAAAAGAGACTCAACAAGAATTACTTGTAAACATAAAAAATCAAAAATCATGATCTTAAAGCATTCACTGAACATCCCACTGGTTTTGTCTAGGACTGAGTGAGAAATATGCAAACCAACAAAGCATTGTTGTTCACTAGGGGGAAAATGACCTAATAAACGCCCGGGGCGTTTATTTAATTTTAGGGATCCAAGCGGAGGCGGTTAATAGATAGGAGGGCTTTTTTATTAAAAGAGAGAGAGAGGGAGACGTTTATTCTCATAACTGTAACAAGCTCAAGAGAACAAATATGCTTTCGGCAAAAATATCAAGAGAGTTTTAAAGTGGTGGAATATCCTGACTCCATCAATTTATACGTCTTGGCAGTCTAGAGATCCATATCACTCTTACAAATCGTAACATCGATGTCAGAATGCTCGCTCAGGGTCATGTGTTGCCATCATTAGTCCACTCTAAAGTTGACATTGAACAAGATGAAATACGCAAAGCCTTGTTAATCAAAGGAGGAAACTAAATGAATTAAAAGATTTTGCTGCTTTTTGCTAATTCCTAGTATTTTGGAGTAATTCTCATAGGGGGCGTCTATTGAAGAGGGGCGTCTAATAAAATTTTCACAGCGTAGAGGGGGCGTTTATTAGATAAGAGGCGTTTATTTGGAAGTGGGAGTTTATTAGGTTTACGGTAGTCTTATTTCATCACTAATTTATAGTTGCACAAATCTGGTTGTACCCTGTTTTGCGTGCGCAAATTTTACCTTAACCCTTAAAATATATTCAGTTGTTCTTGCATCAACGCGAAACCTTTACCGAGTGTAAATATTTTTGATTTTCTTCGTTTTAACCATTCTTTTATTTAAAGTGAATTATCTCCGAAAAGAACTTTTAAAGTGTCTCACCATGTCAAGAAATCCAAGACAGTCTTGAATTCTGGATTCTACGCCGCGGATTCCGGATTCCAGGTACTGGATTCCAGTCTCTTACAGTGGAACTTGGATCGGCTGGATTCCTTGAGTTGTATTCCGGATTCCAAAGCACAGGATTTCGGATTCCGCAAGCAAAACAATTCCCGGATTCTGGAATCCGGATTCCCTTATAAGGGACGAAAAGTGTGAGAGTCTCGTTCCCAGAGGCCGCGATCCTTTTGTACAGCGACGAGGATGGTAGATACTTCATATATGATTCCTATAGTGTACCGATTTGCCTCATCCGCTGGACAAAGGTAACGGAGGCTCTGGGGACGAGACTGAAAGTGTGAGAGTTTCAGATATGCTTAAGCGCAAGATTAGGTGTCATCGGTGGCCTCACGCCAGCACCCCGATAACCTGTATAGATCCTGAATCCAAAAATTTATCAAAGTCAAAACTACTATTTAACACAAAATTACCCATCCCACACAACTAGCACTTTTCAATTCCGATACGGCCTGATGGTTTACACGGAGCTCAAGGTTGATGGTTTTCTGATCCACATGAATTACCTGAGGATGCAATTCGTTAACTAAAACGCGTTTTTACATCCCTCAATAAATAATGTATACTTCTTAATGGAAATTGGAGTTCATATTACGTGTACCGGCAACAAATGGTTTTGTGATGACTGTATCAAATAATTAGATAATTGTCCGGTAGCCGATTAAGAGGTCATATTACCACTGTCCACATCAAAGAGTTCATCAACCCTCGAGAGGTTATTGTTCACTTTGTATTGACGCCAGAAAGAGGGGATACTCAAACACGCGTAAAAGGTCAGTAGTATCTTATTAAGCTGTTATGAAAGCGGAATATACAGATGTACCAACGTTTAATTCAGCCCGGAAAGTCATCACTGAAAATTCGAATTTTACAAAAAAAATTGCTGGATTCATTCCTTAATGGAATGGAGCCTTGAGGTTCGGTTTATATTCGTATTCGGTCAGGGCCACCCAACGATTGTTTTCTGTAAAATATATGTGTGAGGAAGCACATTTTGTCTGGAAATTTTTATAGCTTGAGAAAGGCTTAAGATGATTACCCTATTTCCCTTTCGTATTTGAAAAACACCAGAGTAACCAGAGTTAAGAGTAATTTTACCAGCGCGCATTGGATCTCTCATTGATCGTTTTCACTGAAACGCCTCTCATCCCTGGCCTGCCCATACAGTTTTCAGGCGAAAATTTGCCTAAGCAGTAAGCACCGTAGATATTAGAACGTCTCTTTCTTTTGCTAAGAACTAACATAGCCTGTTGTCGGAAAAAAATGTTGCTGAAATACGTGCTGCTTTTACTTTCCCTGTCTCCTTTAATACTGAACTTTCTGGTTTGCCCTGCATACGCGGCATTGTTGGACAAACCAGCTTGCAATCACTGGGGCCATGTTGCTCGCGTGACTTTCAAGAGTTCAATCATAGGAGAAAACAAGCAGCACTTAAAAAGATATCCAGAGGAATTGCTCAGTTCCGGACATGAAAAATTAAAATCTTAAAGCATTCACTGAACATCCCACCGGTTTTGTTTAGGATTGAGCAATGTGCAAATCAACAAAGCCTTGTTGTTAACAAGGGGGGAAATGATCTAATAAAGGTCCAGGTTAGGGGTCCAAGTGGGGGCGTGTTATAGATAGGAGGCGTTTAAAACAGAGAGGCGTTTATTCTCATAACTGTAACAAGCTCAACAAAGCAAATATGCTTTCGGCGAAAGTATCAAGAGAGTTTAAAATTGGCGGAATTCGAATATCCGCTCCATCAATTGATACGTCTAGGCCGTCTAGAAATCTATATCATTGCACTCTTACATATCCTAACATCGATATCAGACTGCTCGCTTAGGGTGATTGTGTTGCCATCATTAGTCTATCCTCAGTGACTCTAAACTTGACACTGAACAATGCAAGATGAAATAGGCAAATCCTTGTTTGTTAATTGAGGGAGAAAACTAAATGAATGAAAAGATTTTGCAATCCCTACTGAGCCTTTTGGAGTAATTCTCATAAAGGGCGTCAATTAGGGAGGGGCTTCTCATATTTTCACAGCGGAGGGGAGGCGTTTATTTATTTATTTTTGTTTACAGATTAGGTCGATTATCATGTAAGGTTTTTATCGTTGGCCTCCGGCTATCTCTCTCACTGTCAATGGTGATCGCGAGCACCCCGATAACCTATAAAGATGAAATCTGAAACATAACCATCGAGTAAATTCAAACAGGCGTCTCGATTTCTAAATCAAACCCGAAACTATTATATAACACAAAATTACCCATCACTATTCCAATACGGTCTGTTGGTTAACACAGAACTCAAGGTTGATGGTTTTCTGATCCACATGAATTCCCTGTGGATGCAATTCATTAACTAAAAAGCGTTTTTTCATGCCTCAATAAATAATGTATACTTCTTAATGGAAATTGGAGTTCATATTACGTGTACCTGCCACAAATGGTTTTGTGATGACTATATCAAATAATTAGATAATTACCTCAATCAGGAGCTGACCTCAATATAGCCGATTACGAAGTCATATTACCAGTGTCCACATTTAAGATTTCATCAACCCCCTAGAGATTATTGTTCACTTTGTATTGAAGCCAGAAAGCGGGAGGGGATACTCAAACACGCTTAAAGGTTAGTAGTATCTTACTGGTAATAGTTGGCACTACAGCTGCCCCCGCTCTGTATATTGTCGGTTAATAGTATTCTTGCTCGTTATGTAGCTGTTTGAAATAAGCCGATTCATAATGAAGATGTTCACACATAGTCCATACAACAGGTGAGATAAATCTGCGGGGGCAATTAAGCTGTTATGAGGGTGGAATATACAAATGTGCCAACGTTTTATTCAGTCCAGAAAGTCATCACTGGAAATTCGAATTTGGCCACCCGAATAAAATGTTTCGTTTCCCTTCGCCCCGTCTCTTGTGATGGTCGGTCGGTCGGGCAAAAGAAAAGAAAAAAGAATGAACACTTGAAGGGGTCTGCGTTCCAGAGAAGGCCGGGCTGCTAAGTAAGAAAGCCTGGCTACAAAGTCAACTCGAAAACGTGGTCTTTTCGACTATTAAATGTCTAAATGGCCTTTAAAAAACGTAGTATCGATGTTAATTAAAGACAAGGACATCACATCATACCAATTGTTGTTAATAAAACAAGATCGTGTGCTTGTAAATTAAAGTACCCTTTTGAAACTAGAGAAGTCCGAAAATCATTTGGTTTACTTTCGTTCCGTGGTGTGATAACTAATTAAAATTAACCACTGTAATATAACAAAAGCTCGTGATGTGTGTGCATTAGCTATTTGCCTATGGGTAAACGCTGGCTAAAGCTGGCGAGTCCACTTATTGTATCTTCTGAGGCCGTTTCTGAGGAAAAGTCCTTAAACTGTAGCTTAACATAACGATGCCTATATATGGCGAAATTTCGACAGTGGTCCCTGATTGTATGGTTGGTTCTCATTGTGACGTCTTTAGGATACGCCCATATATGACCCAATACAGAGATTCGTGGCAAATGATTCTTAATCACCCAAACTCCATACAGACACCGTCAACTTCACCACCATTTCACAGTTTTCAGTGGTAAAAGTAACTCCTGAATTTAAGCCGGAAGCAATGAGACCAGTTGTTGATTATCTGAAACAAATATTTTCTTTTTTCAGTGCATGACGTTTATTTAGTCAATTCTCCTCTTTATAAAGCAGAGGTGCCCAAAGACGGTTTCCTCCTAAATACATTGAGAACACTCTTTACGTGTCCAGATTACTATTAAGATCTATAGAACCAAGAGAGGATGAAGGGGCCTTTATCCTCTCTTGATAGAACTGAATGCATTCTAAGCGACGCTATGACGTTTGTACCTGTGCGGTCATCCTAGGGGAACTTGAGTCTTGTCGAAATTTCAAGGGCTTCAGTATACATTTTTTGATTCCTCCCTGACTCACCTTCACAGTTTTGAATGCGAAAATTTCAGGTTTCCTTTTTTGAACGAAAAAAAAAAGGCCTAACCTGGGCAGTAAAAAAAACAACAAAATTGTCATTGAAAATCGTAGGGAATTTATTAGAGGCTTCGAAAATTGTACATAAATGGAACGGTCATCTAGATTTTTTTTTTATAAGCGGTTTTCCGAACAAAAATGGTAAACAACAGATGCCGTACATGTACGACATTCTAACTGAATCTATTTTGGTGGCTCCTTAACTTTTAGCCATTAGTTACATATCATTCCATGTTTGTCATCTATTTACATTCGTATACTGTTTTTAAAACCCTTACAGTCTGCTTACGCCGGTTTACTTTTTCCTAATAATTTTGAAAACAAAATTCATTATCAGACTAAGGTTTTACTATAAGGTTTGTTTATAGTGGAAAGTGCACTTAGCTTATCCAGCTAGTTTACAGGCACTCCACTCAAATACTTAGAAACAAATAATTTAAACATAACATAACAGGATTAAAAACCCCACTGGCAGGAGGCAACTAGTTGGCTGTTTACAAGTGTGGCCAAGGATTTCAAATCCAGCAAGTGGTCAGAGCGGGACTCGAACCCAGGACCGCGGGATTGCGAATTTACGCGCTGACCACTCTTCGACATCGGCTTCAGTTTTAAATTCTTATAATTTTACTATCTTAAGATATATTCTATTCGAGAAACGTATGTGTTTCTAACCGAAATATATACATTTCGTATATTCATACGTAGTTTATGTTGCTTTTCCCTCTTGTCTTCACCGCGAGGGATCAATTTGTTGTCTTTTTCCTTTATTTTTCTCAACACGAGAAGCACGAGGACTTGCCGTTGTCTTGGTAAATGATCCTTTTCGGATCTTCCGTTTTTTTGGTAGTGAAGAATCCATATGATCTGAGATCGCAAATCCGTTTTTAGATTCTCCCAAATAGACGCAGAGTCATGTTCAGTGCCGGATCCAGGCCTTGAGATAAGGGTCAGTTCAGAAAAAAATTTATTCGGCCCTTCGAGGCCTCAGCTTGGTCTAAAAATAAGGTGGGGGTGGGGGGGGGGGGGGGGCTGTGGGTAAAAGGTAGGATCCGGTTGTTTTTTTAAAAAAAAAATTGAAAGAAAAAAATAGTCGCCCACTCTTTTGAACTCCAACCCGGTCTTNTAGAATTTTAAAATGAGCTTGGTCAGGGCGGTCATCCTTCTGTTCTCCATTGCGCTGGTAACTCAGTCAATCTTTGGGTCTCCCTTCAAACAAAACAATCATGTCAAAAGGAACCCTGGATCGTCTACTCATGACACTGAGGTAAAATATATTGTCTTTGAAATGAGATACGTACGGTGGGTGTTCAAGTCAATTTTGGTGACTTTCGTTTAAATCGAACATGAAAGTTTCTTTTGGAGGTAATACAGTATGTTGCCCAGTATCCGGACACTTCAGTTTAACATTGCAATAACTTTCCTTTGTTAGTCGCAAGAGCTCTGAAATTTGGCCCAGAGAAAATCTACTTAGTCCTTAATATGACGAAAGACAGTTTAACTTTTTTATCTTTGTTTCCATCGCGAAATTAATATTTTTGCAGAGCTCCTATGTTGCCCAGTATCCGGACAGCTGGCCCAGTGTCCGGACACAACAATAACAACGTAATTGTACATAAATTTCGACAGGCAAGCGACTTATAAGCTTCTCTTGCACTTGCAAAGTAGTCTGGCAATCGATTCCAAAAATATAACCTAGCATCGTGAAATAAAACATAACAAATGACCGGGGTATGGTGGGGAACGCGAGCGGTTGCTAAATGGCATAATTCTCACTTCCTTGTCTAGGAACTTTTTTCCCTGATCATGTAAGGAAGGCATCCATGGACATGCCGAAGCCGCAGTTTCCTAGCTCAATTAGCCAATCTGCAAAAGACTTTTTTTTCCTTCATTTTTTATTTTAAAAAAAAGCTTGCGGAAGGGACCTCAATGCGACAGTCAAAGGTCACTCTCCTATTTATAGTCTGACCTGCGGTGTTGATTTCTTCATGCTCAGTCCCGACAGACCTTACAATAAGCCTGTTTTTCTAGCACTAATTCCTCCTTCTTTCACATCTCTCAATCCCTTTGTGACATTATTTAGACCGTTGCAACCCCATTCTAGCAGTCACAACTGGGGAAAGTATGAGAATTCCAGCTTCAACTCCGACGCTTTTGATTTTTATATGGACAATGCAGTCACGCGAAACAAGCGGCGCACACGAAATCAAGTCGCCTCCGAATGAGTGAAGAAAATTTCCGTACGGAATTGAGTAGAATAACATTTTACGACTACATCATATATCCTAAGCTTTAATTATAGTTATAGATATGAAATAAATCTTATCCGGGTATAGGTATTCATCGATTCTGTACAGCAAAGAAAAAACTGTCCGGATACTGGGCAACCTAACATCAATACTTAGTGAATGTTTTTGGGCTCCGTTATTCCAAACAAATCGAATTTCAAGAAGAATCAATAAAATTTCTGAGAACCTGACTTCTTTAAGTATCTTCACTTTAAATATTAAACCGTTTGTTTGAAAATATCACGTATTACCTACCCTTTTCTGAGCAGCACTAATTTTACCGCGCAGTAAACAGCGTAAATATTAGCCATAAAATGTTTATTCACCTGAACAGAACGGCGTCTACTGCGACTGTTTCGCAAGCTTTCAACTCGCCAAAACCATCATCTTAAAGCTGAATTGTTCAGCTTTCATTCGAAAAACTTCGTTTACCGATAACTGAAAAGGGCGCCTCGAAAATTGAGTTTTTGTTTTAAGAGCCAGTCTCTGTAAGATCCTCATATCGAGTTTTGCCCACAAAATGGATTTCGCTCATAATTTTTCTGTAGACTTCTTTTAATAAGTATATAACAAATATGAAACTAGATTGGAGAAATTCGCTTCTGTAAATTTTTTTTAACGAATTTTCTTTCGGCGCCACATGAGGACCTGAGATGCCTGTGAAATATGCAAATTTTGTCAAAATTCAACCGATTGCTCCGGATCATAGTCCCCTCTACTAACTATGTCCGGATAAAAGAGTTCGACCCAAAGCTTCCAATTTATTAGTTATTTTAATTGAGCCTTTATCTTTACAATAATACAGGTCAGAATCAGCAACACCTTTTCGTTTAGAAAATCTGAGGAAACACACTTAGCCCCTTTGACCCACTTAGCGAAACATACGATTTTAGCCAAATTCAGTGGATTAAATCTCAAAAGTGGCTCACACTTACAGACTCTCCTGCATATCATTGTGTAGTTCAATCCTTCAGCTACTGAATCGTGTTAAAAGTTTTGGCGGCCATTCAGGTTCGGTCGAGGGGTGAGTGGCGAAACTTGCCTTACTTTGCCATTTCGCCGGTGTTTCGCCATCGTGAAGTCCAGAAGGATTGTCAGAATAGAAAGCGAGAAATCGGGTAAATGCCACTCGAAACTTGTCCATTCCTAAAGACTGCAT


gene-Peve_00000001  1382    20  1382    1383
gene-Peve_00000039  2535    1423    2535    2536
gene-Peve_00000112  271 3979    271 272
gene-Peve_00000117  11913   4271    11913   11914
gene-Peve_00000122  35013   16205   35013   35014
gene-Peve_00000008  2351    51239   2351    2352
gene-Peve_00000013  21088   53611   21088   21089
gene-Peve_00000032  7683    74720   7683    7684
gene-Peve_00000035  39241   82424   39241   39242
gene-Peve_00000042  5389    121686  5389    5390

4 Subset Expressed Genes

Only those with at least one read in each sample

4.0.1 Peek at count matrix

head "${count_matrix}" | column -t -s","

echo ""
echo ""

wc -l "${count_matrix}"
gene_id             RNA-POR-71  RNA-POR-73  RNA-POR-76  RNA-POR-79  RNA-POR-82
gene-Peve_00006864  0           0           3           263         79
gene-Peve_00022042  439         396         552         640         286
gene-Peve_00009109  89          181         33          293         1789
gene-Peve_00009108  0           0           0           0           0
gene-Peve_00004359  4598        2449        6571        2745        10812
gene-Peve_00004358  147         159         24          155         153
gene-Peve_00004353  158         62          461         123         143
gene-Peve_00009100  179507      81177       154976      83235       211948
gene-Peve_00009103  187         16          885         106         263


40390 ../output/06.2-Peve-Hisat/gene_count_matrix.csv

4.1 Import count matrix

# Read the data into a data frame
count_matrix_df <- read.csv(count_matrix, header = TRUE)

str(count_matrix_df)
'data.frame':   40389 obs. of  6 variables:
 $ gene_id   : chr  "gene-Peve_00006864" "gene-Peve_00022042" "gene-Peve_00009109" "gene-Peve_00009108" ...
 $ RNA.POR.71: int  0 439 89 0 4598 147 158 179507 187 0 ...
 $ RNA.POR.73: int  0 396 181 0 2449 159 62 81177 16 17 ...
 $ RNA.POR.76: int  3 552 33 0 6571 24 461 154976 885 0 ...
 $ RNA.POR.79: int  263 640 293 0 2745 155 123 83235 106 0 ...
 $ RNA.POR.82: int  79 286 1789 0 10812 153 143 211948 263 21 ...

4.2 Only genes with at least one read per sample

# Filter rows where all values are greater than 0
filtered_count_matrix_df <- count_matrix_df[apply(count_matrix_df > 0, 1, all), ]


str(filtered_count_matrix_df)
'data.frame':   17224 obs. of  6 variables:
 $ gene_id   : chr  "gene-Peve_00022042" "gene-Peve_00009109" "gene-Peve_00004359" "gene-Peve_00004358" ...
 $ RNA.POR.71: int  439 89 4598 147 158 179507 187 1049 484 128 ...
 $ RNA.POR.73: int  396 181 2449 159 62 81177 16 412 15 60 ...
 $ RNA.POR.76: int  552 33 6571 24 461 154976 885 1785 460 420 ...
 $ RNA.POR.79: int  640 293 2745 155 123 83235 106 433 78 118 ...
 $ RNA.POR.82: int  286 1789 10812 153 143 211948 263 1613 151 127 ...

4.3 Subset genes FastA

Only expressed genes

# Get the row names (gene_ids) of the filtered data frame
filtered_gene_ids <- filtered_count_matrix_df$gene_id

fasta <- readDNAStringSet(genes_fasta)

subset_fasta <- fasta[names(fasta) %in% filtered_gene_ids]
writeXStringSet(subset_fasta, genes_subset_fasta)

4.3.1 Peek at genes subset FastA

head "${genes_subset_fasta}"

echo ""
grep "^>" --count "${genes_subset_fasta}"
>gene-Peve_00000013
ATGAGCTTGGTCAGGGCGGTCATCCTTCTGTTCTCCATTGCGCTGGTAACTCAGTCAATCTTTGGGTCTCCCTTCAAACA
AAACAATCATGTCAAAAGGAACCCTGGATCGTCTACTCATGACACTGAGGTAAAATATATTGTCTTTGAAATGAGATACG
TACGGTGGGTGTTCAAGTCAATTTTGGTGACTTTCGTTTAAATCGAACATGAAAGTTTCTTTTGGAGGTAATACAGTATG
TTGCCCAGTATCCGGACACTTCAGTTTAACATTGCAATAACTTTCCTTTGTTAGTCGCAAGAGCTCTGAAATTTGGCCCA
GAGAAAATCTACTTAGTCCTTAATATGACGAAAGACAGTTTAACTTTTTTATCTTTGTTTCCATCGCGAAATTAATATTT
TTGCAGAGCTCCTATGTTGCCCAGTATCCGGACAGCTGGCCCAGTGTCCGGACACAACAATAACAACGTAATTGTACATA
AATTTCGACAGGCAAGCGACTTATAAGCTTCTCTTGCACTTGCAAAGTAGTCTGGCAATCGATTCCAAAAATATAACCTA
GCATCGTGAAATAAAACATAACAAATGACCGGGGTATGGTGGGGAACGCGAGCGGTTGCTAAATGGCATAATTCTCACTT
CCTTGTCTAGGAACTTTTTTCCCTGATCATGTAAGGAAGGCATCCATGGACATGCCGAAGCCGCAGTTTCCTAGCTCAAT

17224

5 BLASTx

5.1 Download SwissProt

cd "${blastdbs_dir}"

curl -O https://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/uniprot_sprot.fasta.gz

mv uniprot_sprot.fasta.gz 20250618-uniprot_sprot.fasta.gz

gunzip --keep 20250618-uniprot_sprot.fasta.gz
  % Total    % Received % Xferd  Average Speed   Time    Time     Time  Current
                                 Dload  Upload   Total   Spent    Left  Speed
  0     0    0     0    0     0      0      0 --:--:-- --:--:-- --:--:--     0  0 88.7M    0  374k    0     0   547k      0  0:02:46 --:--:--  0:02:46  546k 27 88.7M   27 24.7M    0     0  14.5M      0  0:00:06  0:00:01  0:00:05 14.5M 65 88.7M   65 58.1M    0     0  21.7M      0  0:00:04  0:00:02  0:00:02 21.7M 94 88.7M   94 83.6M    0     0  22.6M      0  0:00:03  0:00:03 --:--:-- 22.6M100 88.7M  100 88.7M    0     0  22.7M      0  0:00:03  0:00:03 --:--:-- 22.7M

5.2 Create BLASTdb

cd "${blastdbs_dir}"

"${diamond}" makedb \
--in 20250618-uniprot_sprot.fasta.gz \
--db "${diamond_db}" \
--quiet \
--threads "${threads}"

5.3 Run DIAMOND BLASTx

"${diamond}" blastx \
--db "${blastdbs_dir}"/"${diamond_db}.dmnd" \
--query "${genes_subset_fasta}" \
--out "${diamond_output}" \
--outfmt 6  \
--sensitive \
--evalue 1e-10 \
--max-target-seqs 1 \
--block-size 15.0 \
--index-chunks 4 \
--threads "${threads}" \
2> "${output_dir}"/diamond-blastx.log

head "${diamond_output}"

echo ""

wc -l "${diamond_output}"
gene-Peve_00000013  sp|P58691|ACTPA_RADCR   42.0    112 57  4   20741   21073   69  173 1.43e-15    81.6
gene-Peve_00000035  sp|Q9NBX4|RTXE_DROME    24.9    486 325 16  10287   11693   428 890 2.49e-17    95.5
gene-Peve_00000042  sp|P62993|GRB2_HUMAN    51.8    56  27  0   887 720 161 216 3.81e-11    68.2
gene-Peve_00000058  sp|A3KN95|T151B_XENTR   29.2    202 135 5   1732    2322    127 325 1.16e-12    75.5
gene-Peve_00000107  sp|F4IE66|PRP22_ARATH   64.5    76  27  0   10104   9877    41  116 1.08e-20    103
gene-Peve_00000108  sp|Q6P4S8|INT1_MOUSE    51.7    89  42  1   5664    5930    180 267 3.17e-20    105
gene-Peve_00000114  sp|F1R8Z9|FXJ1B_DANRE   73.1    67  14  1   2663    2475    183 249 5.64e-21    102
gene-Peve_00000115  sp|Q9DCV4|RMD1_MOUSE    33.3    147 85  4   8274    7870    71  216 3.40e-13    76.6
gene-Peve_00000116  sp|Q32KL4|RMD1_BOVIN    47.6    84  42  1   8369    8124    85  168 2.34e-12    74.3
gene-Peve_00000121  sp|O50655|XERD_SELRU    25.5    314 209 10  25765   26682   29  325 2.56e-13    79.3

8966 ../output/30.00-Peve-transcriptome-GOslims/Pevermanni-expressed-genes.blastx.outfmt6

6 GENE ONTOLOGY

6.1 Get gene IDs and SwissProt IDs

awk -F"|" '{print $1"\t"$2}' "${diamond_output}" \
| awk '{print $1"\t"$3}' \
> "${output_dir}"/gene-SPIDs.txt

head "${output_dir}"/gene-SPIDs.txt

echo ""
echo ""

wc -l "${output_dir}"/gene-SPIDs.txt
gene-Peve_00000013  P58691
gene-Peve_00000035  Q9NBX4
gene-Peve_00000042  P62993
gene-Peve_00000058  A3KN95
gene-Peve_00000107  F4IE66
gene-Peve_00000108  Q6P4S8
gene-Peve_00000114  F1R8Z9
gene-Peve_00000115  Q9DCV4
gene-Peve_00000116  Q32KL4
gene-Peve_00000121  O50655


8966 ../output/30.00-Peve-transcriptome-GOslims/gene-SPIDs.txt

6.2 Get SwissProt IDs

awk -F"|" '{print $2}' "${diamond_output}" \
| sort --unique \
> "${output_dir}"/SPIDs.txt

head "${output_dir}"/SPIDs.txt

echo ""
echo ""

wc -l "${output_dir}"/SPIDs.txt
A0A096X8J7
A0A0C2SRU0
A0A0G2JZ79
A0A0G2L7I0
A0A0P0XB70
A0A0R3K2G2
A0A0R4I9Y1
A0A0R4IBK5
A0A0R4IES7
A0A0R4ITC5


6137 ../output/30.00-Peve-transcriptome-GOslims/SPIDs.txt

6.3 Retrieve UniProt records

A difference in number of records could be due to retrieval from only “reviewed” records, while BLAST may have included both “reviewed” and “unreviewed.” SwissProt records

python3 \
../../M-multi-species/code/uniprot-retrieval.py \
"${output_dir}"/SPIDs.txt \
"${output_dir}"/

gunzip "${output_dir}"/uniprot-retrieval.tsv.gz

echo ""
echo ""

wc -l "${output_dir}"/uniprot-retrieval.tsv
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
500 / 500
137 / 137


6149 ../output/30.00-Peve-transcriptome-GOslims/uniprot-retrieval.tsv

6.4 Map GO to Genes

# Read gene-SPIDs.txt
gene_spids <- read.delim(file.path(output_dir, "gene-SPIDs.txt"), header = FALSE, stringsAsFactors = FALSE)

colnames(gene_spids) <- c("GeneID", "Entry")

# Read uniprot-retrieval.tsv
uniprot <- read.delim(
  file.path(output_dir, "uniprot-retrieval.tsv"),
  header = TRUE,
  stringsAsFactors = FALSE,
  check.names = FALSE
)

# Merge on Entry
gene_SPID_GOID_merged <- merge(gene_spids, uniprot[, c("Entry", "Gene Ontology IDs")], by = "Entry", all.x = TRUE)

# View result
str(gene_SPID_GOID_merged)
'data.frame':   8966 obs. of  3 variables:
 $ Entry            : chr  "A0A096X8J7" "A0A096X8J7" "A0A0C2SRU0" "A0A0C2SRU0" ...
 $ GeneID           : chr  "gene-Peve_00003282" "gene-Peve_00027371" "gene-Peve_00011467" "gene-Peve_00011913" ...
 $ Gene Ontology IDs: chr  "GO:0005452; GO:0005886; GO:0008509; GO:0015701; GO:0051453; GO:0055085; GO:0086001" "GO:0005452; GO:0005886; GO:0008509; GO:0015701; GO:0051453; GO:0055085; GO:0086001" "GO:0004609; GO:0005739; GO:0006646" "GO:0004609; GO:0005739; GO:0006646" ...

6.4.1 Write merged to file

# Optionally, write to file
write.table(gene_SPID_GOID_merged, 
            file = file.path(output_dir, "gene-SPIDs-GOIDs.tsv"), 
            sep = "\t", 
            row.names = FALSE, 
            quote = FALSE)

6.5 Clean up merged

full.gene.df <- as.data.frame(gene_SPID_GOID_merged)

# Clean whitespace, filter NA/empty rows, select columns, and split GO terms using column name variables
gene.GO.df <- full.gene.df %>%
  mutate(!!"Gene Ontology IDs" := str_replace_all(.data[["Gene Ontology IDs"]], "\\s*;\\s*", ";")) %>% # Clean up spaces around ";"
  filter(!is.na(.data[["GeneID"]]) & !is.na(.data[["Gene Ontology IDs"]]) & .data[["Gene Ontology IDs"]] != "") %>% 
  dplyr::select(all_of(c("GeneID", "Gene Ontology IDs")))


str(gene.GO.df)
'data.frame':   8781 obs. of  2 variables:
 $ GeneID           : chr  "gene-Peve_00003282" "gene-Peve_00027371" "gene-Peve_00011467" "gene-Peve_00011913" ...
 $ Gene Ontology IDs: chr  "GO:0005452;GO:0005886;GO:0008509;GO:0015701;GO:0051453;GO:0055085;GO:0086001" "GO:0005452;GO:0005886;GO:0008509;GO:0015701;GO:0051453;GO:0055085;GO:0086001" "GO:0004609;GO:0005739;GO:0006646" "GO:0004609;GO:0005739;GO:0006646" ...

6.6 Flatten gene GOID file

flat.gene.GO.df <- gene.GO.df %>% separate_rows(!!sym("Gene Ontology IDs"), sep = ";")

str(flat.gene.GO.df)
tibble [106,348 × 2] (S3: tbl_df/tbl/data.frame)
 $ GeneID           : chr [1:106348] "gene-Peve_00003282" "gene-Peve_00003282" "gene-Peve_00003282" "gene-Peve_00003282" ...
 $ Gene Ontology IDs: chr [1:106348] "GO:0005452" "GO:0005886" "GO:0008509" "GO:0015701" ...

6.7 Group by GOID

grouped.gene.GO.df <- flat.gene.GO.df %>%
  group_by(!!sym("Gene Ontology IDs")) %>%
  summarise(!!"GeneID" := paste(.data[["GeneID"]], collapse = ","))

str(grouped.gene.GO.df)
tibble [12,132 × 2] (S3: tbl_df/tbl/data.frame)
 $ Gene Ontology IDs: chr [1:12132] "GO:0000002" "GO:0000009" "GO:0000012" "GO:0000014" ...
 $ GeneID           : chr [1:12132] "gene-Peve_00012113,gene-Peve_00027057,gene-Peve_00036001,gene-Peve_00043435,gene-Peve_00020141,gene-Peve_000383"| __truncated__ "gene-Peve_00020421" "gene-Peve_00036938,gene-Peve_00028780,gene-Peve_00013011,gene-Peve_00032733" "gene-Peve_00023146,gene-Peve_00032460,gene-Peve_00008105,gene-Peve_00013484,gene-Peve_00013490,gene-Peve_000098"| __truncated__ ...

6.8 Vectorize GOIDs

# Vector of GO IDs
go_ids <- grouped.gene.GO.df[["Gene Ontology IDs"]]

str(go_ids)
 chr [1:12132] "GO:0000002" "GO:0000009" "GO:0000012" "GO:0000014" ...

6.9 Prepare GOslim OBO

# Find GSEAbase installation location
gseabase_location <- find.package("GSEABase")

# Load path to GOslim OBO file
goslim_obo_destintation <- file.path(gseabase_location, "extdata", goslims_obo, fsep = "/")

# Download the GOslim OBO file
download.file(url = goslims_url, destfile = goslim_obo_destintation)

# Loads package files
gseabase_files <- system.file("extdata", goslims_obo, package="GSEABase")

6.10 GOslims from OBO

# Create GSEAbase GOCollection using `go_ids`
myCollection <- GOCollection(go_ids)

# Retrieve GOslims from GO OBO file set
slim <- getOBOCollection(gseabase_files)

str(slim)
Formal class 'OBOCollection' [package "GSEABase"] with 7 slots
  ..@ .stanza     :'data.frame':    153 obs. of  1 variable:
  .. ..$ value: chr [1:153] "Root" "Term" "Term" "Term" ...
  ..@ .subset     :'data.frame':    22 obs. of  1 variable:
  .. ..$ value: chr [1:22] "Rhea list of ChEBI terms representing the major species at pH 7.3." "Term not to be used for direct annotation" "Terms planned for obsoletion" "AGR slim" ...
  ..@ .kv         :'data.frame':    2110 obs. of  3 variables:
  .. ..$ stanza_id: chr [1:2110] ".__Root__" ".__Root__" ".__Root__" ".__Root__" ...
  .. ..$ key      : chr [1:2110] "format-version" "data-version" "synonymtypedef" "synonymtypedef" ...
  .. ..$ value    : chr [1:2110] "1.2" "go/releases/2025-06-01/subsets/goslim_generic.owl" "syngo_official_label \"label approved by the SynGO project\"" "systematic_synonym \"Systematic synonym\" EXACT" ...
  ..@ evidenceCode: chr [1:26] "EXP" "IDA" "IPI" "IMP" ...
  ..@ ontology    : chr NA
  ..@ ids         : chr [1:141] "GO:0000228" "GO:0000278" "GO:0000910" "GO:0001618" ...
  ..@ type        : chr "OBO"

6.11 Biological Process GOslims

# Retrieve Biological Process (BP) GOslims
slimdf <- goSlim(myCollection, slim, "BP", verbose)

str(slimdf)
'data.frame':   72 obs. of  3 variables:
 $ Count  : int  86 19 19 417 51 85 20 4 88 55 ...
 $ Percent: num  1.077 0.238 0.238 5.224 0.639 ...
 $ Term   : chr  "mitotic cell cycle" "cytokinesis" "cytoplasmic translation" "immune system process" ...

6.12 Map GO to GOslims

# List of GOslims and all GO IDs from `go_ids`
gomap <- as.list(GOBPOFFSPRING[rownames(slimdf)])

# Maps `go_ids` to matching GOslims
mapped <- lapply(gomap, intersect, ids(myCollection))

# Append all mapped GO IDs to `slimdf`
# `sapply` needed to apply paste() to create semi-colon delimited values
slimdf$GO.IDs <- sapply(lapply(gomap, intersect, ids(myCollection)), paste, collapse=";")

# Remove "character(0) string from "GO.IDs" column
slimdf$GO.IDs[slimdf$GO.IDs == "character(0)"] <- ""

# Add self-matching GOIDs to "GO.IDs" column, if not present
for (go_id in go_ids) {
  # Check if the go_id is present in the row names
  if (go_id %in% rownames(slimdf)) {
    # Check if the go_id is not present in the GO.IDs column
    # Also removes white space "trimws()" and converts all to upper case to handle
    # any weird, "invisible" formatting issues.
    if (!go_id %in% trimws(toupper(strsplit(slimdf[go_id, "GO.IDs"], ";")[[1]]))) {
      # Append the go_id to the GO.IDs column with a semi-colon separator
      if (length(slimdf$GO.IDs) > 0 && nchar(slimdf$GO.IDs[nrow(slimdf)]) > 0) {
        slimdf[go_id, "GO.IDs"] <- paste0(slimdf[go_id, "GO.IDs"], "; ", go_id)
      } else {
        slimdf[go_id, "GO.IDs"] <- go_id
      }
    }
  }
}

str(slimdf)
'data.frame':   72 obs. of  4 variables:
 $ Count  : int  86 19 19 417 51 85 20 4 88 55 ...
 $ Percent: num  1.077 0.238 0.238 5.224 0.639 ...
 $ Term   : chr  "mitotic cell cycle" "cytokinesis" "cytoplasmic translation" "immune system process" ...
 $ GO.IDs : chr  "GO:0000022;GO:0000070;GO:0000082;GO:0000086;GO:0000132;GO:0000281;GO:0007052;GO:0007064;GO:0007076;GO:0007079;G"| __truncated__ "GO:0000281;GO:0000915;GO:0007110;GO:0007111;GO:0007112;GO:0032465;GO:0032466;GO:0032467;GO:0036089;GO:0036090;G"| __truncated__ "GO:0001731;GO:0001732;GO:0002182;GO:0002183;GO:0002184;GO:0002188;GO:0002191;GO:0017183;GO:0140708;GO:1900248;G"| __truncated__ "GO:0001771;GO:0001774;GO:0001776;GO:0001777;GO:0001779;GO:0001780;GO:0001782;GO:0001805;GO:0001807;GO:0001812;G"| __truncated__ ...

6.13 Flatten GOslims

# "Flatten" file so each row is single GO ID with corresponding GOslim
# rownames_to_column needed to retain row name info
slimdf_separated <- as.data.frame(slimdf %>%
  rownames_to_column('GOslim') %>%
  separate_rows(GO.IDs, sep = ";"))

# Group by unique GO ID
grouped_slimdf <- slimdf_separated %>%
  filter(!is.na(GO.IDs) & GO.IDs != "") %>%
  group_by(GO.IDs) %>%
  summarize(GOslim = paste(GOslim, collapse = ";"),
            Term = paste(Term, collapse = ";"))


str(grouped_slimdf)
tibble [5,531 × 3] (S3: tbl_df/tbl/data.frame)
 $ GO.IDs: chr [1:5531] " GO:0000278" " GO:0002181" " GO:0002376" " GO:0003014" ...
 $ GOslim: chr [1:5531] "GO:0000278" "GO:0002181" "GO:0002376" "GO:0003014" ...
 $ Term  : chr [1:5531] "mitotic cell cycle" "cytoplasmic translation" "immune system process" "renal system process" ...

6.14 Counts of GOslims

slimdf.sorted <- slimdf %>% arrange(desc(Count))

slim.count.df <- slimdf.sorted %>% 
  dplyr::select(Term, Count, Percent)

str(slim.count.df)
'data.frame':   72 obs. of  3 variables:
 $ Term   : chr  "anatomical structure development" "signaling" "cell differentiation" "immune system process" ...
 $ Count  : int  1696 727 714 417 273 257 243 206 198 186 ...
 $ Percent: num  21.25 9.11 8.94 5.22 3.42 ...

6.14.1 Write GOslims to file

Need to create a column name for GOslimIDs from data frame rownames.

# Create header vector
header <- c("GOslimID", colnames(slim.count.df))

# Write header to file
writeLines(paste(header, collapse = "\t"), 
           file.path(output_dir, "GOslim-counts.tsv"))

# Append data frame contents to existing file, which contains header info
write.table(
  slim.count.df,
  file = file.path(output_dir, "GOslim-counts.tsv"),
  sep = "\t",
  row.names = TRUE,
  quote = FALSE,
  col.names = FALSE,
  append = TRUE
)

RESULTS

Multispecies barplots were created of the top 10 GOslims with the highest GO term counts.

Multispecies barplots of top 10 GOslims by GO term counts.

Plots were generated with the following code:

# Define file paths and species names
files <- list(
  "D-Apul" = "~/gitrepos/urol-e5/deep-dive-expression/D-Apul/output/30.00-Apul-transcriptome-GOslims/GOslim-counts.tsv",
  "E-Peve" = "~/gitrepos/urol-e5/deep-dive-expression/E-Peve/output/30.00-Peve-transcriptome-GOslims/GOslim-counts.tsv",
  "F-Ptuh" = "~/gitrepos/urol-e5/deep-dive-expression/F-Ptuh/output/30.00-Ptua-transcriptome-GOslims/GOslim-counts.tsv"
)

# Read and combine data
goslim_data <- map2_dfr(
  files,
  names(files),
  ~ read_tsv(.x, show_col_types = FALSE) %>%
    mutate(Species = .y)
)


# Find the top 10 Terms by Count for each species
top_terms <- goslim_data %>%
  group_by(Species) %>%
  slice_max(order_by = Count, n = 10, with_ties = FALSE) %>%
  ungroup() %>%
  distinct(Term)

# Filter the data to only include these Terms
goslim_top <- goslim_data %>%
  filter(Term %in% top_terms$Term)

# Plot: Term on Y, Count on X
ggplot(goslim_top, aes(y = fct_reorder(Term, Count), x = Count, fill = Species)) +
  geom_bar(stat = "identity", position = position_dodge(width = 0.8)) +
  labs(
    title = "Top GOslim Term Counts by Species",
    y = "GOslim Term",
    x = "Count"
  ) +
  theme_bw() +
  theme(
    axis.text.y = element_text(size = 8),
    plot.title = element_text(hjust = 0.5)
  )

7 REFERENCES

Buchfink, Benjamin, Klaus Reuter, and Hajk-Georg Drost. 2021. “Sensitive Protein Alignments at Tree-of-Life Scale Using DIAMOND.” Nature Methods 18 (4): 366–68. https://doi.org/10.1038/s41592-021-01101-x.
Carlson, Marc. 2017. “GO.db.” https://doi.org/10.18129/B9.BIOC.GO.DB.
Danecek, Petr, James K Bonfield, Jennifer Liddle, John Marshall, Valeriu Ohan, Martin O Pollard, Andrew Whitwham, et al. 2021. “Twelve Years of SAMtools and BCFtools.” GigaScience 10 (2). https://doi.org/10.1093/gigascience/giab008.
H. Pagès, P. Aboyoun. 2017. “Biostrings.” https://doi.org/10.18129/B9.BIOC.BIOSTRINGS.
Martin Morgan, Seth Falcon. 2017. “GSEABase.” https://doi.org/10.18129/B9.BIOC.GSEABASE.
Quinlan, Aaron R., and Ira M. Hall. 2010. “BEDTools: A Flexible Suite of Utilities for Comparing Genomic Features.” Bioinformatics 26 (6): 841–42. https://doi.org/10.1093/bioinformatics/btq033.