Matt:LabNotes/2014-5-14

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Designing Hexamer Oligo Primers for RT - Enriching mRNA Rolonies[edit]

Method[edit]

  • Download mRNA, rRNA, and MT-rRNA sequences from biomart in fasta format
    • Ensembl Genes 75 Database
    • Homo sapiens genes (GRCh37.p13) Dataset
    • Filter with Gene type: protein_coding, rRNA, MT-rRNA
      • 92,169 protein-coding transcripts
      • 568 rRNA transcripts
    • Attributes: Sequences of Unspliced (Transcript)
  • Use RNAhexamerDistribution.pl to count number of instances each of the 4096 hexamer sequences appeared
    • Output is a \n separated list of length 4096
      • The index of the list corresponds to hexamer sequence using a quaternary numeral system where A = 0, T = 1, C = 2, G = 3
        • e.g. AAAAAA = 000000 = 0 (or the first number in the list)
        • e.g. AAAAAT = 000001 = 1 (or the second number)
        • e.g. AAGAAA = 001000 = 192 (or the 193rd number)
  • 64 hexamers with 10,000+ occurrences in mRNA and 0 in rRNA
  • Use MakeHexamerKey.pl to generate a list of hexamer sequences (converts integers 0-4095 to hexamer)

Method 2: Exon Sequences[edit]

  • Same as original process but select "Exon sequences" for Attributes

Compare to sequences of Genes of Interest[edit]

  • Same as Method 2 (Exon sequences only) but select list of Entrez IDs instead of protein_coding
    • Got Entrez_IDs by getting genes from Agi26k0gap ppDesigner Output and Agi26k20gap ppDesigner Output and filling most Entrez IDs from ppDesigner TargetFile
      • Had to manually fill in entrez IDs that didn't exist in TargetFile_combined.txt
      • After removing duplicate genes between 0gap and 20gap, left with 3,999 Entrez Gene IDs
  • Used Biomart to get Exon sequences of those 3,999 genes
  • Used RNAhexamerDistribution.pl on fasta file
    • 97 hexamers with occurences in targeted genes exons and 0 in rRNA (exons)

Order[edit]

  • Cost: $10 for 5' Phosph + $0.18/base (24 bases)
    • 97 primers = ~$1,400
    • Order only half (48) with the highest number of occurrences in targeted mRNA

Sequences Ordered, Genes, and Hexamer Key