Matt:LabNotes/2014-2-3

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BLAST RT RAB7A primer sequences against rRNA[edit]

BLAST with ribosomal RNA[edit]

  • Used Biomart sequence retrieval tool, searched for unspliced (transcript) and used Filter = Gene: rRNA with Associated Gene Name as heading for fasta file
    • Downloaded and stored in my genome-miner as RNAsequences.fa in /RT_RAB7A
  • Built database
 formatdb -i RNAsequences.fa -p F
  • Saved a fasta file of RT primer sequences and then BLAST-ed with wordsize of 7, 8, 9, and 10
 blastall -i RT_RAB7A_primers.txt -d RNAsequences.fa -p blastn -o result_wordsize7.txt -W 7
 blastall -i RT_RAB7A_primers.txt -d RNAsequences.fa -p blastn -o result_wordsize8.txt -W 8
 ...
  • Shortened primer sequences to 7 by removing from the 5'end and BLAST-ed with wordsize of 7
 blastall -i primers_7nt.txt -d RNAsequences.fa -p blastn -o result_7.txt -W 7

Results[edit]

Conclusion[edit]

  • Looking only at primers that match at the 3' end, the only alignments are to RNA pseudogenes
    • 1000nt RT primer has NO alignments of any kind
  • Therefore it is unlikely that it is because of high amounts of rRNA primary rolonies that 1000nt shows so much signal