Draft: Part 2 of Lab 2 Methods

The Basic Local Alignment Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website was used to find expressed sequence tags (ESTs) that match the sequence of our unknown gene. A Nucleotide BLAST of the unknown sequence was performed, setting the database to “expressed sequence tag (est)” and the organism to “Brachypodium distachyon.” The sequences that had an Identity number of at least 95%, meaning they perfectly or near perfectly matched cDNAs, were selected and saved in a new file as “Unknown-AHP-ESTs-fasta.txt.” Consensus sequences (contig) of the ESTs were formed using the CAP3 web server at the Pasteur Institute. The contigs were saved in a new text file named “AHP-CAP3-Contigs,” and the sequences of ESTs that were not contig’ed were saved in another text file named “AHP-CAP3-SingleSequences.” A full-length cDNA of the unknown sequence was then found by performing a Nucleotide BLAST with the “Nucleotide collection (nr/nt)’ database instead. Once a cDNA sequence for the gene was found, it was compared to the contigs from CAP3 through a Nucleotide BLAST. The cDNA sequence was pasted as the query sequence, the contigs were pasted as the subject sequence, and the “Align two or more sequences” box was selected. The resulting comparison was saved as a new file named “AHP-Contig+cDNA-fasta.txt.”