Graphic Display in GCG

2. Graphic Display in GCG Configuring Graphics Languages and Devices Program CodonPreference DotPlot Figure Frames FrameSearch /PLOt GapShow GrowTree HelicalWheel Isoelectric MapPlot Moment PepPlot PileUp PAUPDisplay PlasmidMap PlotFold PlotSimilarity PlotStructure PlotTest PrettyBox Prime StatPlot TestCode WordSearch -PLOt • GIF (Graphics Interchange Format) – GIF87a, GIF89a • HPGL (HP Graphics Language) – • ColorPro, HP7470, HP7475, HP7550, HP7580, LaserJet3 • PNG (Portable Network Graphics) – • For WWW Browser • PostScript • ReGIS • Sixel • Tektronix • Xwindows – Dowload x-win412.exe

3. Exercise 08-1 Configuring X-windows Download x-win412.exe from ftp://163.25.92.42 Double click x-win412.exe, accept all default settings. Start x-win32 Connect to GCG via TELNET gcg 2% go gcg 3% xwindows Use XWindows graphics with what device: Color Workstation Gray Scale Workstation Monochrome Workstation Please choose one ( * COLORWORKSTATION * ) Plotting Configuration set to: Language: xwindows Device: COLORWORKSTATION Port or Queue: GCG_Graphics gcg 4% plottest GIF & PostScript

4. Stemloop Identifying Inverted Repeats Mfold Calculating RNA Folding Displaying of Folding Structures Plotfold/Dotplot Nucleic Acid Secondary Structure Stemloop and Mfold In Nucleic acids, inverted repeat sequences may indicate foldback (self pairing)structures.

5. STEMLOOP StemLoop finds stems (inverted repeats) within a sequence. You specify the minimum stem length (number of nucleotides in a paired stretch), minimum and maximum loop sizes, and the minimum number of bonds per stem (length of nucleotide sequence between the paired regions). Vertical bars ('|') indicating the base pairs. The associated loop is shown to the right of the stem. If either the stem or loop is too long to be displayed in its entirety on the line, then only that part that fits on the line is shown. The first and last coordinates of the stem are displayed on the left, and the length of the stem (size), the number of bonds in the stem (quality), and the loop size are shown on the right. start size • 217 AGGCTGCAGTG AGCCGTGAT 11, 25 • |||||| |||| C • 257 TCCGGCCTCAC GTCACCGCG quality end stem

6. 1) See the stems 2) See the stem coordinates 3) File the stems (*.fld) 4) File the stems as points for DOTPLOT 5) Choose new parameters 6) Get a different sequence Sort stems by: 1) Position 2) Quality 3) Size STEMLOOP Output formats • 221 TGCAGTG AGCCGTG 7, 18 • ||||||| • 248 ACGTCAC CGCGCTA 14 • Loop Start End Size Quality • 1 35 54 8 18 • *.stem • *.pnt  DOTPLOT

7. MFOLD • Using energy minimization criteria, any predicted "optimal" secondary structure for an RNA or DNA molecule depends on the model of folding and the specific folding energies used to calculate that structure. Different optimal foldings may be calculated if the folding energies are changed even slightly. Because of uncertainties in the folding model and the folding energies, the "correct" folding may not be the "optimal" folding determined by the program. You may therefore want to view many optimal and suboptimal structures within a few percent of the minimum energy. You can use the variation among these structures to determine which regions of the secondary structure you can predict reliably. For instance, a region of the RNA molecule containing the same helix in most calculated optimal and suboptimal secondary structures may be more reliably predicted than other regions with greater variation. Mfold output file: *.mfold

8. Survey of optimal and suboptimal foldings A) sub-optimal energy plot B) p-num plot Sampling of optimal and suboptimal foldings C) circles D) domes E) mountains F) squiggles PLOTFOLD MFOLD How to read *.mfold?

9. PLOTFOLD A) sub-optimal energy plot

10. PLOTFOLD B) p-num plot

11. PLOTFOLD plotC) circles

12. PLOTFOLD D) domes

13. PLOTFOLD E) mountains

14. PLOTFOLD F) squiggles

15. Exercise 08-2 Stemloop & X-windows Open the file “exercise08-2.doc” and follow the steps. gcg2 4% fetch gb:d00063 d00063.gb_pl1 gcg2 5% stemloop d00063.gb_pl1 There are 16 stems. Would you like to 1) See the stems 2) See the stem coordinates 3) File the stems 4) File the stems as points for DOTPLOT 5) Choose new parameters 6) Get a different sequence Q)uit? Please choose one (* 1 *): Try 1-4 Sort stems by: 1) Position 2) Quality 3) Size Q)uit Please choose one (* 1 *):

16. Exercise 08-3 Mfold & Plotfold Open the file “Exercixe08-3.doc” and follow the steps. gcg2 4% fetch gb:j02061 J02061.gb_vi gcg2 5% mfold j02061.gb_vi  j02061.mfold $ Mfold (Linear) MFOLD what sequence ? j02061.gb_vi Begin (* 1 *) ? End (* 121 *) ? What should I call the energy matrix output file (* j02061.mfold *) ?

17. Primer Selection Specificity - %GC - Dimer – Hairpin - Tm Amino Acid sequences Nucleotide sequences Pileup Pretty Prettybox CONSENSUS Nucleotide Amino Acid backtranslate Primer Selection Program-Prime Confirm by BLAST

18. Primer Length Minimum - Maximum- ----------------------------------------------PCR Product Length Minimum - Maximum- ----------------------------------------------Maximum number of primers or PCR products in output (range 1 thru 2500) Primer DNA concentration (nM) (range .1 thru 500.0) - Salt concentration (mM) (range .1 thru 500.0) - ----------------------------------------------Select: forward primers, only reverse primers, only primers on both strands for PCR Set maximum overlap (in base pairs) between predicted PCR products Forward strand primer extension must include position Reverse strand primer extension must include position ---------------------------------------------- Reject duplicate primer binding sites on template Specify primer 3' clamp (using IUB ambiguity codes) ----------------------------------------------- Primer % G+C Minimum (range 0.0 thru 100.0) Maximum -----------------------------------------------Primer Melting Temperature (degrees Celsius) Minimum (range 0.0 thru 200.0) Maximum -----------------------------------------------Maximum difference between melting temperatures of two primers in PCR (degrees Celsius)(range 0.0 thru 25.0) ----------------------------------------------- Product % G+C Minimum (range 0.0 thru 100.0) Maximum ----------------------------------------------- Product Melting Temperature (degrees Celsius) Minimum (range 0.0 thru 200.0) Maximum

19. Exercise 08-4 Primer Selection Use the human npm cDNA sequence you obtained in Exercise06 as query to design a pair of primers that will copy the whole coding sequence when translated in frame. THEN Check the specificity of the primers by using BLAST.