A. Identify OrgaNism

1. Grow organism in pure culture.

2. PCR amplify 16s rRNA gene with universal primers 27f & 1492r. Sanger sequence with 27f to identify, use 5 primers to assemble complete 16s sequence and deposit in GenBank.

3. BLAST search to identify organism. If full sequence is <98.7% identical to a published type strain, organism is novel! If <99.5% identical and equidistant from several species, organism is likely to be novel. Construct NJ Tree in MEGA.

B. Sequence Genome

1. Isolate gDNA, PCR amplify 16s rRNA gene from gDNA with universal primers 27f & 1492r. Sanger sequence with 27f to confirm correct DNA.

2. Combine with other samples on a shared NextGen Sequencing Run at a core sequencing facility. Target is 50-100x coverage.
   a. PacBio is best
   b. Illumina MiSeq (2 x 300b preferred)

3. Assemble genome using PATRIC.

4. Upload assembly to RAST for annotation, confirm 16s rRNA sequence by BLAST.

5. Deposit WGS assembly into GenBank and raw reads into SRA archive at NCBI

C. Identify Closest Relatives

1. a. Best method: After genome sequence is incorporated into RefSeq, wait for Genome Taxonomy DataBase (GTDB) tree to be updated.
   b. Download GTDB tree, open in NCBI Genome Workbench, use accession to search for correct branch in complete tree, create subtree, export as Newick.
   c. Open subtree in MEGA, search NCBI to determine organism names from accessions, label branches in MEGA to determine organisms with most recent common ancestor.

2. a. Use RAST to retrieve rpoB gene sequence, BLASTn vs RefSeq and WGS databases restricting to family of closest relatives as identified by 16s rRNA gene sequence.
   b. Download best match sequences, make ML tree to determine organisms with most recent common ancestor.

3. Prepare Overall Genome Relatedness Index (OGRI) Table with at least 10 entries (and the type species of genus)
   a. BLAST genome-derived 16S rRNA vs 1. RefSeq Genomes; 2. 16S rRNA (fill in high % matches missing from 1); 3. nr/nt nucleotide collection (fill in high % matches missing from 1 & 2); 4. Add organisms clustering with novel organism on GTDB or rpoB trees above
   b. Download best match genomes in fasta (save) and GenBank (upload to RAST) formats
   c. Calculate eDDH using GGDC, ANI using OAT, AAI with RAST Sequence Based Comparison Tool & NewmanLab AAI Calculator

D. CulTure Collections

1. Deposit organism into two culture collections in different countries. Some are easier to work with than others. I recommend BCCM/LMG, KCTC, DSMZ.

2. Literature research on closest relatives - focus on IJSEM, SAM, AvL papers

3. Order/Request needed reference strains from culture collections.

E. Analyze Genome Sequence

1. Prepare Venn Diagram to identify unique and shared genes

2. Generate hypotheses based on gene content

3. Identify genes corresponding to traits identified in experiments below

F. Phenotyping

1. Examine colony and cell morphology (Gram stain, wet mount)

2. Test for growth under different environmental conditions - temperature, pH, [NaCl], oxygen availability.

3. Assess antibiotic sensitivity and resistance via Kirby-Bauer assay.

4. Perform assays for common enzymes such as catalase, oxidase, tryptophanase, amylase, caseinase, DNase

5. Test for growth on differential and selective media

6. Test responses in Biolog GenIII plates or Biomerieux API strips.

G. ChemoTaxonomy

1. Analyze Fatty Acid Methyl Esters (FAME) using Microbial ID Inc Sherlock System and standard method.

2. Analyze Respiratory Quinones by primary TLC separation, followed by RP-HPLC separation to determine number of isoprene units

3. Analyze Polar Lipids by 2D TLC followed by spraying to detect specific classes of polar lipids

4. Analyze Pigments - Acetone extracts separated by HPLC with a Diode Array Detector to obtain 3D plots and UV-Vis spectra of specific peaks