deletions | additions       diff --git a/Organism Identification using 16s rRNA gene sequence.md b/Organism Identification using 16s rRNA gene sequence.md  index 6fbc6e2..dd7f8d0 100644  --- a/Organism Identification using 16s rRNA gene sequence.md  +++ b/Organism Identification using 16s rRNA gene sequence.md 
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It is necessary to screen the 16S rDNA Sanger sequencing results for possible genome sequencing candidates. We recommend starting with BLAST results, then continuing onto the Genomes Online Database (GOLD). This is a large database containing most sequenced genomes and many ongoing sequencing projects. Sometimes the use of GOLD and an internet search will be sufficient to obtain information about the organism you have isolated. In many cases, it will be useful to build a phylogenetic tree to aid in identification, as the BLAST search results may not be sufficiently informative.  ##BLAST 16S rDNA sequence  Begin by navigating to the Standard Nucleotide [BLAST BLAST  at NCBI](http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) NCBI: [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome](http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)  Paste in your Sanger consensus sequence. We recommend checking the box to exclude Uncultured/environmental sample sequences, since these will not be informative for identification. Be sure the nucleotide collection (nr/nt) is selected under database and click the BLAST button. 
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3. Another possibility is that you will get significantly less than 99% identity to any sequences in the NCBI database. One explanation for this is that your sequence is of poor quality. This might require more stringent trimming using SeqTrace or even resequencing if the quality is poor enough to make assigning taxonomy difficult. Another possibility is that you have isolated something that is not very closely related to anything in the NCBI database. In the latter case, we would recommend first re-doing the BLAST search, but unchecking the "Uncultured/environmental sample" to see if the sequence matches others that have been found, but are not associated with a cultured organism. In either case, we would recommend re-sequencing for confirmation and then proceeding to section 11 "Building a 16S rDNA Phylogenetic Tree" to examine the phylogenetic context of the novel sequence.  ##Using GOLD (the Genomes Online Database)  Go to: http://genomesonline.org/cgi-bin/GOLD/index.cgi [http://genomesonline.org/cgi-bin/GOLD/index.cgi](http://genomesonline.org/cgi-bin/GOLD/index.cgi)  Under the Search tab, click the "Quick Search" option and you should be taken to a page that looks like the screen shot displayed in Figure \ref{fig:GOLD}. 
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##Align 16S Sequences using Align Sequences Nucleotide BLAST  First locate the 16S sequences of the genome you'd like to compare to, by searching the NCBI Nucleotide database for "Species 16s gene".  http://www.ncbi.nlm.nih.gov/nuccore/ [http://www.ncbi.nlm.nih.gov/nuccore/](http://www.ncbi.nlm.nih.gov/nuccore/)  Click on the sequence of interest, then click on the "FASTA" link to get the sequence in FASTA format. Now navigate to the ["Align Align  Sequences Nucleotide BLAST" page](http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq) page:  [http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq](http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq)  Paste in the two 16S rDNA sequences and click on the "BLAST" button. Unless both your sequence and the sequence to which you are comparing were amplified with the same primers, the query coverage will not be 100%. A low identity can be the result of poor sequence quality or taxonomic distance.