EvoPrinterHD
EvoPrinterHD Instructions

The fastest/easiest way to get the most out of EvoPrinterHD is to start EvoPrinting with the sample sequence provided below. Special instructions for EvoPrint analysis for bacteria are available. Many of the important steps in the procedure and data interpretations are highlighted by its analysis. The example sequence covers 2 kb of intergenic DNA flanking the Drosophila melanogaster fushi tarazu (ftz) gene and it spans 2 highly conserved regions that are most likely present in all Drosophilids. By following the steps listed below, you will discover that while both are conserved they have rearranged relative to each other in more evolutionary distant species. The key to optimizing the EvoPrinting process is to introduce the more evolutionary distant species one at a time to detect loss of alignment co-linearity due to rearrangements. Conserved sequence blocks within the rearranged DNA are subsequently identified when the second or third alignments for each species is included in the EvoPrint process.

  1. Start by copy/pasting the 2 kb ftz sequence into the Drosophila EvoPrinterHD input window and selecting the "Launch eBLAT alignments" option. (Click here to copy/paste sample sequence.)
  2. When the alignments have been completed, select "Check Input DNA for Repetitive Sequences" option. Single-copy repeat sequences are shown as blue-colored bases and multi-copy repeats are displayed in red-colored. If multi-copy repeats are detected, for the initial analysis consider generating EvoPrints with just the flanking sequences as the conserved sequences within the repeat may interfere with the alignments. The readout for the ftz example indicates that there are no interfering repeats in this sequence.
  3. Next, select the "View Alignment Results" option. In addition to showing alignment scores, first and last aligning input DNA bases and the presence of sequencing gaps, the alignment scorecard contains links to the eBLAT alignments and to composite eBLATs. The composite eBLAT identifies those conserved sequences within the input DNA that have undergone rearrangements and/or duplications in the aligning regions of the different species included in the analysis. High alignment scores in the 2nd and 3rd eBLAT alignments indicate that at least two rearrangements have occurred relative to the reference genome. For example, composite eBLATs reveal that rearrangements containing clusters of conserved sequences are detected in the D. grimshawi, D. mojavensis, D. virilis and D. willistoni genomes.
  4. After viewing the composite eBLATs, select the Continue option. The next scorecard contains quantitative information concerning the extent of conserved sequence rearrangement and/or duplication (R/D values). It also contains selection options for the different alignments that will be used to generate an EvoPrint. By default, the algorithm automatically pre-selects the 6 species closest in homology to the input reference DNA and then deselects those that have sequencing gap(s) in their highest scoring alignment region. For the initial EvoPrint, use the pre-select composite eBLATs. The EvoPrint readout highlights the D. melanogaster sequences that are conserved (uppercase black bases) in all of the pre-selected test species (D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae and D. pseudoobscura). The EvoDifferences profile reveals those sequences that are present in all test species except one. The EvoDifferences for this analysis also indicates that all species share alignment co-linearity (indicated by an even distribution of unique base changes).
  5. Return to the alignment scorecard and select the next species All option. Generate the EvoPrint and EvoDifferences profile readouts and examine the EvoDifferences profile as above. Continue to add species one at a time and examine their contribution to the EvoDifferences profile. Also include those species that have sequencing gaps one at a time to determine if the gap(s) disrupt the analysis.

    The final EvoDifferences profile of the ftz example sequence that includes all of the Drosophila species in the analysis is shown below.

    ftz Genomic Evodifferences Profile - Relaxed EvoPrint

    Black capital letters represent bases conserved in all species and colored bases represent sequences present in all species except D.simulans, D.sechellia, D.erecta, D.yakuba, D.ananassae, D.pseudoobscura, D.persimilis, D.grimshawi, D.mojavensis, D.virilis or D.willistoni.

    ttcgcaaaatctggtttgtagaatgtaaacaattatttgctccaatctgatgaattatgttctcaccgatccaac
    tggaaatatatatccttgcattgcttccagactcctgatcacagatgccgctattcgttagtgaagtacgtagca
    ttttcctggcaaaaccattaaagtacgatataaatttgaaacaaatgccaacttcaaacatatttaaatgtttca
    agggggcggacatccccagctgAGCTGTCAAAtcgAaCAcCGACCTTGGCCATTAATCgTccCATTCAtGTCAAA
    TTTGgACAATTCgAATGTCAATgGAGAAAGTTTtcagccagcctccgacgcgtcagcaGAAAAATCAATAATTC
    gAACGCGTGAATTtCCATTAACAATTTGcTTtGgCGAACTTGAAACGTgaagcacacaaaacAcgagaAGCCTTG
    AAacgacgcTAAAAAATAAATTAGAAAATTCCtccgcacccgccGgCACATGTGCAagagaacccagtgaaattt
    tccacctccattgccgtggagctgaggccatcgactccgctgctcagaattagggttttcatggggtgtggaggg
    caaaatctgtgaggcggggatgttggcgccttgccttatttgagtacatgcaggaggaggattttcagtGTGTct
    gaAAATGgAAATTctgtgtgcgattcgctcACTGaCTGACTGACACATATTTGGGCATGATCAAAATTAATGACC
    CaGCgCTcatataggcccatcaactcgagcacagagacaccttggcacacacactagactgatgcaacatacgaa
    ctgaatttgggtcaatggctcacgtttttttttttttcggtgaatcttttttctaaaacgtaagttttcttcaaa
    tataaaaataactagccatgtttctattacaacacatcttataagccacatataatacttttagacaaagtaaaa
    acttaaatggaaaacaaatatattttgtattgatttgacgtcagtctaaaacaggaagaatcataagaaatagca
    ccaagagcttttaattcatcactctcgttgttcattgtatattgtatataattgtctataaacggcttggttatg
    gtttgtgggaagctaagtaagcttctctgcatcacaacgctcaataattcctgaaatcagcataaaaggtgggat
    tgcagacagccccaaaagcaaaccacccaacgatcgtatctgcaccgcccttgtgcaaatatcatttcgcggatt
    cttttgcagccgtttagcgtcacatgcaatttgccatcattccgagctaaacgctctTTTTgAcCCGaGCTGTCG
    GTTATTAAAAGtCATAATTGAATTTCTTGtAAACAGTTGTTgGTTGTGAAAGATGTGGCCcGAACATGCAAAAtc
    gcagaaataaggggctgaagggtataggatgGggaATCCgGcCTGGCGCTgAAATTTCGAAAGTATGAAAAAAGC
    AATTAAAACTGAAATAATGAATTCTTTCTTTtGCcagtctctggcaTGTTtctcgttcgaggttggagttgaatt
    cggagtcggagttggctggctggctgccttgctGGTgAAGGGATtggccttcgtttcacccatcaccgccagaaa
    atttcccGGGCTGATTAGCATGAGGCGTAACAGTTTTTAATTGCAGCCACATTTcgccgagctccgaatgtgcag
    caacaaaagcctggcaacacataatgatagcaatACTTATCTTGCCATCAGAAAGTcGAAAAAgTTtaagcactt
    tttcaaTTTTTTttgTAAcAAccaccaatgcttctatgcgacagctttccctctactgtgacttctttagatgtt
    ttcggctggattgatatattttatttcgtttaaaagattgttaaaagaaaactAAACTAGAACTGAggcAcaTTT
    TTTGTgtgcTTTATTTTTTctagttcacgggattatttttcgacgatttt
     
    75
    150
    225
    300
    375
    450
    525
    600
    675
    750
    825
    900
    975
    1050
    1125
    1200
    1275
    1350
    1425
    1500
    1575
    1650
    1725
    1800
    1875
    1950
    2025


    Return to EvoPrinterHD home.

[ National Institutes of Health (NIH) | Contact NINDS ]
[ Home | Disclaimer | Privacy Notice | Accessibility Compliance ]
[ National Institute of Neurological Disorders and Stroke (NINDS) | FirstGov | Department of Health and Human Services ]


H H S Logo - link to U. S. Department of Health and Human Services     N I H logo - link to U. S. National Institutes of Health    N I N D S logo - link to National Institute of Neurological Disorders and Stroke    FirstGov Logo - link To FirstGov