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

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Importing Confidence Scores Many base callers generate confidence values associated with each base call. Sequencher supports confidence values from PHRED, ACE and Trace Tuner files, as well as SCF, ABI 3730 and ESD files. Importing PHRED called data Sequencher can read in the SCF, FASTA and Qual files generated by PHRED and supports PHRED base calls and confidence values. To view the confidence levels, you must first set the user preferences for displaying confidence values (See Chapter 21 “Customizing Sequencher and User Preferences” for more information). Then go to the View menu and choose Display Base Confidences. To import the data into Sequencher, it must first be organized in a folder containing three subfolders. These subfolders are chromat_dir which contains the trace files, edit_dir which contains the text files and phd_dir which contains the quality scores. Select File then go to Import and then Folder of Sequences to import the phd_dir folder. To import the sequences you would like to view you may also go to the File menu and choose the Import command with the Sequences... submenu. Importing PHRAP Files Sequencher can import the ACE projects created by PHRAP. To import an ACE project select File then go to Import and then the ACE Project… submenu. Browse to a file that ends with “.ace” or “.ace.x” (such as .ace.1,.ace.2, etc.). This file will typically be located in the subfolder “edit_dir.” Select the highest numbered ACE file to account for all editing and reassembling. Sequencher automatically imports and incorporates as much auxiliary data as it can find. The auxiliary files must be in the same folder as the ACE file or arranged in the traditional PHRAP assembly hierarchy (chromatogram files in a chromat_dir folder and PHD files in a phd_dir folder). If the .singlets file, the .qual file, the PHD files and the chromatogram files all exist and are available, Sequencher will import them. Alternatively, you can import parts of the PHRAP project by going to the File menu and choosing Import and then Sequences or by going to the File menu and clicking on Import and then Folder of Sequences. • If you import the chromatogram files from the "chromat_dir", these fragments will have PHRED base calls and chromatogram data but no base qualities. • If the "_.phd" files are loaded and "_. fasta.qual" files are available, you will have imported all of the fragments from the PHRAP project. They will have base quality and chromatogram data. • The.qual files alone will not load your sequences. • If you import PHD files from the "phd_dir" and the corresponding chromatogram files are available, these files will import with PHRED base calls, chromatogram data and base qualities. SEQUENCHER 4.8 User Manual for Windows © 1991 - 2007 Gene Codes Corporation, Inc. All rights reserved. 24
Note: When using ftp to transfer your data to your local computer, it is ALWAYS important to transfer these files in binary mode. If you are not familiar with ftp, speak with your network administrator. Removing sequences and contigs from a project To remove items from the project, select the items you want to remove. Go to the Edit menu and choose Remove From Project…, or click the Trash Can button in the button bar. Removing an item from a project is permanent, so Sequencher asks whether you really want to do it. Note: Remember, you cannot back out of this action with a simple Undo command, but if you close the project without saving, all changes including removed items will be forgotten. The Revert to Saved Project command has the same effect. Closing an editor window When you have finished entering and/or editing a sequence, click the close button in the top right corner of the window at the right end of the title bar (see Figure 4-4) or choose Close Window from the File menu. Sequencher then removes the Sequence Editor window from the screen. Once you have closed the window, a sequencing icon will represent your sequence. (Figure 4-4). Figure 4-4 Clicking the close box Closing a project If you have finished working with the project but not with Sequencher, choose Close Project from the File menu. Sequencher will ask if you want to save the changes to your project. Click Yes to save your changes and close the project. Click No to close the project without saving your changes. Click Cancel if you wish to continue working with the project. You can then start working with another project by going to the File menu and choosing New Project or Open Project. SEQUENCHER 4.8 User Manual for Windows © 1991 - 2007 Gene Codes Corporation, Inc. All rights reserved. 25
Page 2 and 3: THIS IS AN AGREEMENT BETWEEN YOU, T
Page 4 and 5: © 1991 - 2007 Gene Codes Corporati
Page 6 and 7: Templates .........................
Page 8 and 9: Set Circular Genome Size ..........
Page 10 and 11: The Bases View ....................
Page 12 and 13: Protein translations for the Consen
Page 14 and 15: Setting header, footer and margin o
Page 16 and 17: Page Intentionally Left Blank xv
Page 18 and 19: We welcome your comments or support
Page 20 and 21: Navigating Sequencher Help The Sequ
Page 22 and 23: Contig menu The commands in the Con
Page 24 and 25: Page Intentionally Left Blank SEQUE
Page 26 and 27: Figure 3-1 An empty Sequencher Proj
Page 28 and 29: Figure 3-4 Project Window with Larg
Page 30 and 31: value in the Confidence pane of the
Page 32 and 33: Editing the information for a seque
Page 36 and 37: How to import using menu commands I
Page 38 and 39: Figure 4-2 Browse for Folder dialog
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 80 and 81: Chapter 9 “Sequence Assembly” f
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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When you use a Name Delimiter, it w
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If the Expression is a delimiter bo
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Figure 10-6 The Project Window with
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Page Intentionally Left Blank SEQUE
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it is in the reverse orientation. T
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Figure 11-2 Contig overview Fifure
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Base Numbers At Transitions Clickin
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Label Name Function E F G H Sequenc
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Consensus to Forensic Standards In
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consensus is from a base with a low
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move the gaps to the 5’ end, sele
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Figure 12-4 Fill Void dialog box De
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First select the base or range of b
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The Translated Variance Table If yo
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Structure of the Translated Varianc
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(see Figure 13-5). It is possible f
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Figure 13-7 Translated Variance Tab
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If you want to restrict the Compari
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Figure 13-13 Creating a Joined feat
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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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