Genbank - Curto Results File

• Took master file BLAST-combined.fasta and needed to filter out curto sequences
• Build algorithm to sort through file and pull out curto sequences and write in new file
• Make a reference text file with curto accession numbers
• curto-accession-numbers.txt
• Run code to make new curto only file
• curto-only2.py
• Problem - new file had >4000 sequences (should be 982)
• Multiple duplicate accession numbers - need to remove
• Build algorithm to keep unique accession numbers
• duplicate-removal.py
• All curto and frigo bacteria -> curto-and-frigo-only.fasta
• Added outlier sequence (AB695377.1 Sediminihabitans luteus) for phylo reference
• curto-and-frigo-only-with-outlier.fasta

• Create OTUs within curto and frigo genera 
• Used QIIME pick_otus.py
• Use default confidence intervals (97% (n = 41))
• Use curto-and-frigo-only-with-outlier.fasta as input file
• curto-and-frigo-only-with-outlier_otus.txt
• Generated biom file - not important with such closely related taxon
• otu-table.biom
• Pick representative sequence for each OTU
• pick_rep_set.py
• rep_set.fna 
• Assign Taxonomy to each rep set to make sure everything has worked so far
• assign_taxonomy.py
• /taxonomy-results/rep_set_tax_assignments.txt
• Must align multiple rep sequences to template - greengenes core database (16S gene)
• align_seqs.py
• Use PYNAST with min length of 75% of the median sequence length
• Filter alignment (filter_alignment.py)
• Remove positions which are gaps in every sequence (common for PyNAST, as typical sequences cover only 200-400 bases, and they are being aligned against the full 16S gene)
• Removed some OTUs due to failure to align (moved to new file):
• OTU2
• OTU3
• OTU4
• OTU8
• OTU10
• OTU11
• OTU26
• OTU30
• OTU37
• OTU38
• OTU39
• OTU40
• Removal of these OTUs reduced the overall number of samples by 50
• Should have 933 samples left in 30 OTUs
• Make phylogeny - newick file 
• make_phylogeny.py
• Need to reroot file (in FigTree) for outlier

For Reference: QIIME Review