EvoPrinting is accomplished in six steps:

• 1. Curate the reference-DNA FASTA sequence (up to 25 kb) from FlyBase or Ensembl.
• 2. Paste sequence into the BLAT input window at UCSC and generate the pairwise alignment with the desired genome.
• 3. Copy-paste the BLAT aligned reference-DNA output (upper sequence) into one of the EvoPrinter input boxes (without removing spaces or numbering) and then enter the test-genome identity in the box flanking the input window.
  Look at the BLAT output of the 'test species' to see if there are sequence gaps (indicated by Ns). If a test species has multiple gaps, consider excluding it from the EvoPrint or check that species with EvoDif to determine if the sequence gap removes MCSs that are present in other species.
• 4. Select additional species at the BLAT alignment engine and repeat step 3 without removing the input reference-DNA
• 5. After all of the desired BLAT readouts have been loaded into different EvoPrinter windows, generate the EvoPrint. Different EvoPrints can be generated from subsets of the selected species by deselecting one or more species and then generating another EvoPrint.
• 6. To generate an EvoDifference Print, select just one species and then select the 'Find Differences' button.In addition, one can exclude species by checking the appropriate box.

• 1. Make multiple prints selecting different species. EvoPrinter provides information about evolutionarily variable sequences as well as conserved sequences. In addition EvoPrints generated from less diverged species may pick up additional important MCSs.
• 2. Use the EvoDif feature to understand what individual species contribute to the final EvoPrint.
• 3. Regulatory sequences may not be positionally conserved between evolutionarily distant species, nor are they always found in a large enough conserved context to be detected byEvoPrinter. However many of these elements can be identified when evolutionarily distant species are excluded from the EvoPrinter analysis. Thus it is important to generate multiple EvoPrints using different combinations of species.

• 1. The curated FASTA sequence from the database is copy-pasted into Microsoft Word. This maintains the gene annotation found in the database.
• 2. Copy-paste the EvoPrint readouts on a new page of the Microsoft Word document.
• 3. Convert the font in this document to Courier and reduce font size to 8.
• 4. For large EvoPrints, to maximize nucleotides seen on a single page, remove all carriage returns and increase margin width.
• 5. To highlight conserved MCSs, convert the non-conserved nucleotides (lower case letters) from black to dark gray by using the replace tool in Microsoft Word and convert the whole document to bold face text.
• 6. Highlight the different gene structures (UTRs and coding sequence) with background colors. For example, see Figures 1 and 2 in Odenwald et al., 2005.

A step-by-step EvoPrinting example using the vertebrate Mash-1 CNS enhancer is provided.

Discover MCSs using EvoPrinter