Guided tour:

1. Suppose you are interested in Hepatitis B Virus NCBI GenBank accession number AB014366. The first step is to obtain a set of related sequences. You could do this via the NCBI blast site. Alternatively, for viruses, you could use the NCBI viral taxonomy databases.
   
   
2. Align the sequences and obtain a phylogenetic tree pairs file here. Alternatively, you could use e.g. PHYLIP to calculate a phylogenetic tree and convert it a pairs file here.
   
   
3. Annotate the known CDSs in the reference sequence. E.g. for AB014366 you can use the NCBI GenBank annotation (the CDS lines). Be aware that the GenBank annotation is sometimes incomplete.
   
   
4. Now you are ready to go to the MLOGD base page here.
   
   
5. If you have a specific query CDS (e.g. a long ORF that has not previously been annotated as a CDS, but that you suspect may be a CDS) then use the 'Test input query CDSs' option. Input the query CDS location and click on 'Calculate'. The results page gives:
   
   
   • Null versus alternate model likelihood ratio statistics, where the null model is that the query ORF is not coding while the alternate model is that the query ORF is coding (both the null and alternate models include the annotated CDSs).
     
     
   • A plot of the likelihood ratio statistics (example plot). You may follow a link here to generate simulated sequences under the same null and alternate models. The simulations are used to put error bars on the likelihood ratio statistics, and to interpret the synonymous/nonsynonymous and N1/N2/N3 statistics (example plot).
     
     
   • A nucleotide-by-nucleotide plot of the likelihood ratio statistic for each reference - non-reference sequence pair and summed over the phylogenetic tree (example plot). Gaps and stop and start codons for each sequence are also annotated on the plot. You may follow a link here to zoom in on the plot, add grid lines, or adjust the running-mean window size. There is also a zoomed in version of this plot (example plot).
     
     
6. If you don't have a specific query CDS, you may either select the 'Find and test all non-annotated ORFs' option, or select the 'Six-frame sliding window plots' option.
   
   
7. The 'Find and test all non-annotated ORFs' option will find all non-annotated ORFs and produce the same statistics and plots as the 'Test input query CDSs' option for each of these ORFs.
   
   
8. The 'Six-frame sliding window plots' option will calculate the MLOGD statistic in a window sliding along the alignment in all six reading frames (example plot). Extended regions of positive signal may indicate potential new CDSs, especially where there is an absense of stop codons. Once you have located any potentially interesting regions, you can then go back to the 'Test input query CDSs' option to investigate these regions in more detail.