Sequencher 4.8 User Manual--PC - Bioinformatics and Biological ...

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Chapter 9 “Sequence Assembly” for more information). There are numerous other options under the Assembly Parameters and the Contig menu. Setting the Assembly Conditions Setting assembly parameters To change the parameter settings, select the Project Window. Then either click the Assembly Parameters button in the Project Window or go to the Contig menu and choose the Assembly Parameters command. Sequencher displays the dialog box shown in Figure 9-1. Figure 9-1 Assembly parameters dialog box Sequencher uses the values set in this window to control the way it assembles sequences. Once you have set the options you require click the OK button to dismiss the Assembly Parameters window. Assembly Algorithms To choose which assembly algorithm you want to use, click on the Assembly Parameters button on the Project Window and then click on one of the three radio buttons at the top of the window. Your choice will depend on your data. SEQUENCHER 4.8 User Manual for Windows © 1991 - 2007 Gene Codes Corporation, Inc. All rights reserved. 64
For instance if you click the Clean Data radio button, contig assembly is faster but Sequencher may miss some possible matches if the ends of your sequences contain a large number of ambiguities. For best performance, make sure that you have trimmed poor quality data from your sequences. The Dirty Data algorithm is somewhat slower because Sequencher is performing more rigorous comparisons between the sequences. You should note that automated sequencers create dirty data and Sequencher’s algorithm is optimized to deal with that. The Large Gap algorithm allows you to assemble sequences that are expected to contain insertions and deletions (gaps) greater than 10 bases long. Such gaps are typically found in evolutionary studies and sequence comparisons from different organisms. The Large Gap algorithm is similar to and slower than the dirty data algorithm but allows larger gaps to occur when performing the assembly. Typical examples of assemblies that require the Large Gap algorithm include the comparisons of a cDNA sequence to a genomic sequence and the assembly of related genes with alternative splicing. Assembling with Dirty Data Go to the Assembly Parameters dialog box and select the Dirty Data radio button. There are two sliders for changing minimum match values. To set the proportion of bases that have to match use the Minimum Match Percentage slider. To set the minimum number of bases that must overlap use the Minimum Overlap slider. Change the settings by positioning your cursor on the slider, holding the mouse button down and dragging the slider left or right. The value set will automatically update as you move the slider. For example, if you attempt to assemble a 17-base sequence that matches perfectly to a selected 1000 base sequence with the minimum overlap set at 20 bases (Sequencher’s default minimum overlap), the two fragments will not assemble. You must first reduce minimum overlap to 17 bases or lower and then assemble the fragments. Assembling with Clean Data When you select the Clean Data algorithm, an additional option called Maximum Loop Out Size is available. This has an elevator button for adjusting size value (Figure 9-2 below). This parameter is the largest number of consecutively mismatched bases that are acceptable in a potential overlap. Change the setting by positioning your cursor on the appropriate arrowhead, and holding the mouse button down. The maximum value, which can be set, is 6. To set the proportion of bases that have to match use the Minimum Match Percentage slider. To set the minimum number of bases that must overlap use the Minimum Overlap slider. SEQUENCHER 4.8 User Manual for Windows © 1991 - 2007 Gene Codes Corporation, Inc. All rights reserved. 65
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THIS IS AN AGREEMENT BETWEEN YOU, T
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© 1991 - 2007 Gene Codes Corporati
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Templates .........................
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Set Circular Genome Size ..........
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The Bases View ....................
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Protein translations for the Consen
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Setting header, footer and margin o
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We welcome your comments or support
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Navigating Sequencher Help The Sequ
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Contig menu The commands in the Con
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Page Intentionally Left Blank SEQUE
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Figure 3-1 An empty Sequencher Proj
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Figure 3-4 Project Window with Larg
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Page 32 and 33: Editing the information for a seque
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Page 36 and 37: How to import using menu commands I
Page 38 and 39: Figure 4-2 Browse for Folder dialog
Page 40 and 41: Importing Confidence Scores Many ba
Page 42 and 43: If you have finished working with S
Page 44 and 45: Selecting sequences Selecting seque
Page 46 and 47: Renaming a sequence or contig Renam
Page 48 and 49: Closing the Project Window When you
Page 50 and 51: Performing a trim with default sett
Page 52 and 53: additional control within the Trim
Page 54 and 55: Figure 6-7 The polylinker window No
Page 56 and 57: Setting Vector Trimming Criteria Se
Page 60 and 61: Basic Reference Sequence properties
Page 62 and 63: When the To Reference by Name comma
Page 66 and 67: A locked editor is read-only. You c
Page 68 and 69: If your sequence was called “MyPU
Page 70 and 71: Viewing summary information in the
Page 72 and 73: Figure 8-7 ORF selected by Next Met
Page 74 and 75: If you want to assign a personal an
Page 76 and 77: Voice verification Sequencher provi
Page 82 and 83: Figure 9-2 The Clean Data algorithm
Page 84 and 85: Performing the assembly Automatic a
Page 86 and 87: Figure 9-5 Interactive assembly sho
Page 88 and 89: The new contig will appear in the P
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Page 92 and 93: If the Expression is a delimiter bo
Page 94 and 95: Figure 10-6 The Project Window with
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Page 100 and 101: Figure 11-2 Contig overview Fifure
Page 102 and 103: Base Numbers At Transitions Clickin
Page 104 and 105: Label Name Function E F G H Sequenc
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Page 108 and 109: consensus is from a base with a low
Page 110 and 111: move the gaps to the 5’ end, sele
Page 112 and 113: Figure 12-4 Fill Void dialog box De
Page 114 and 115: First select the base or range of b
Page 118 and 119: The Translated Variance Table If yo
Page 120 and 121: Structure of the Translated Varianc
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Page 124 and 125: Figure 13-7 Translated Variance Tab
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In either case, the method you choo
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You can edit your data directly in
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Figure 13-17 The Translated Varianc
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Figure 13-19 Extract from Variance
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The Population Table lists the vari
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If you want to remove sample sequen
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Select Individual Variance Reports
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If you prefer to copy your Translat
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You can copy your Translated Varian
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Summary report display options Sequ
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Enabling the protein display To ena
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Note: Remember that even if a seque
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If you select the Contig Chromatogr
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Select command Table 15-1 Summary o
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Note: The Extend Selection command
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Figure 16-2 Restriction map options
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example, if you were displaying onl
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Figure 16-8 Checkmarked enzyme list
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display this view by going to the V
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The default setting shows exact mat
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Creating and editing features To cr
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Quick feature creation To create a
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Figure 18-1 A dimmed icon Finding t
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Note: If you pause between characte
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a dialog box (Figure 18-6). This le
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Figure 19-1 Export Options dialog b
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You should remember to provide a re
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Figure 19-3 Copy As Protein Transla
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[Project Name] and [User Name]. You
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Colors as Backgrounds Sometimes, di
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Choosing a genetic code You will fi
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time you select the Review button f
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Label & Name The Label & Name prefe
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Click the Use Blank Project radio b
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Use the Columns For Multiple Traces
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If you are making changes to the wa
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Variance Table The Variance Table p
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Figure 22-1 The Validate mtDNA Prof
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Figure 22-3 The Circular Genome Siz
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Menu/Window Command Windows Select
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You may require a more complex deli
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Code Base(s) Meaning 5 A or Another
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Feature Display Color Style Second
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eferences numbers, sequences, or us
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Qualifier type Description Citation
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Feature Key Default Feature Qualifi
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Name CursorProject Window Dialog bo
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