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The Staden Package Manual Last Update on 22 October 2002 The Staden Package Manual Last update on 22 October 2002 James Bonfield, Kathryn Beal, Mark Jordan, Yaping Cheng and Rodger Staden Copyright c 1999-2002, Medical Research Council, Laboratory of Molecular Biology. Permission is given to duplicate this manual in both paper and electronic forms. If you wish to charge more than the duplication costs, or wish to make edits to the manual, please contact the authors by email to [email protected]. i Short Contents 1 Sequence assembly and finishing using Gap4.................... 3 2 Searching for point mutations using pregap4 and gap4 .......... 207 3 Preparing readings for assembly using pregap4 ................ 223 4 Marking poor quality and vector segments of readings .......... 291 5 Screening Against Vector Sequences ........................ 293 6 Screening Readings for Contaminant Sequences ............... 305 7 Viewing and editing trace data using trev .................... 309 8 Analysing and comparing sequences using spin ................ 319 9 User Interface ......................................... 401 10 File Formats .......................................... 411 11 Man Pages ........................................... 445 References ............................................... 477 General Index ............................................. 479 File Index ................................................ 491 Variable Index ............................................ 493 Function Index ............................................ 495 ii The Staden Package Manual iii Table of Contents Preface ............................................................. 1 1 Sequence assembly and finishing using Gap4 ...... 3 1.1 Organisation of the gap4 Manual ................................ 3 1.2 Introduction ................................................... 3 1.2.1 Summary of the Files used and the Preprocessing Steps... 5 1.2.2 Summary of Gap4’s Functions .......................... 7 1.2.3 Introduction to the gap4 User Interface.................. 8 1.2.3.1 Introduction to the Contig Selector ............. 9 1.2.3.2 Introduction to the Contig Comparator ........ 10 1.2.3.3 Introduction to the Template Display.......... 12 1.2.3.4 Introduction to the Consistency Display ....... 14 1.2.3.5 Introduction to the Restriction Enzyme Map ... 16 1.2.3.6 Introduction to the Stop Codon Map .......... 17 1.2.3.7 Introduction to the Contig Editor ............. 18 1.2.3.8 Introduction to the Contig Joining Editor ...... 21 1.2.4 Gap4 Menus ......................................... 22 1.2.4.1 Gap4 File menu.............................. 22 1.2.4.2 Gap4 Edit menu ............................. 22 1.2.4.3 Gap4 View menu ............................ 22 1.2.4.4 Gap4 Options menu .......................... 23 1.2.4.5 Gap4 Experiments menu ..................... 23 1.2.4.6 Gap4 Lists menu ............................. 23 1.2.4.7 Gap4 Assembly menu ........................ 23 1.2.5 The use of numerical estimates of base calling accuracy .. 25 1.2.6 Use of the "hidden" poor quality data.................. 27 1.2.7 Annotating and masking readings and contigs........... 28 1.2.7.1 Standard tag types ........................... 28 1.2.7.2 Active tags and masking...................... 28 1.3 Contig Selector ............................................... 30 1.3.1 Selecting Contigs ..................................... 30 1.3.2 Changing the Contig Order............................ 31 1.3.3 The Contig Selector Menus ............................ 32 1.4 Contig Comparator............................................ 33 1.4.1 Examining Results and Using Them to Select Commands ....................................................... 34 1.4.2 Automatic Match Navigation .......................... 35 1.5 Contig Overviews ............................................. 36 1.5.1 Template Display ..................................... 36 1.5.1.1 Reading and Template Plot ................... 38 1.5.1.2 Reading and Template Plot Display ........... 38 1.5.1.3 Reading and Template Plot Options ........... 41 1.5.1.4 Reading and Template Plot Operations ........ 42 1.5.1.5 Quality Plot ................................. 43 1.5.1.6 Restriction Enzyme Plot...................... 45 1.5.2 Consistency Display .................................. 45 1.5.2.1 Confidence Values Graph ..................... 47 1.5.2.2 Reading Coverage Histogram.................. 47 1.5.2.3 Read-Pair Coverage Histogram ................ 48 1.5.2.4 Strand Coverage ............................. 49 1.5.3 Plotting Consensus Quality............................ 51 1.5.3.1 Examining the Quality Plot................... 51 iv The Staden Package Manual 1.5.4 Plotting Stop Codons ................................. 53 1.5.4.1 Examining the Plot .......................... 53 1.5.4.2 Updating the Plot............................ 53 1.5.5 Plotting Restriction Enzymes .......................... 54 1.5.5.1 Selecting Enzymes ........................... 54 1.5.5.2 Examining the Plot .......................... 54 1.5.5.3 Reconfiguring the Plot ....................... 55 1.5.5.4 Creating Tags for Cut Sites ................... 55 1.5.5.5 Textual Outputs ............................. 55 1.6 Editing in Gap4............................................... 57 1.6.1 Moving the visible segment of the contig................ 59 1.6.2 Names ............................................... 60 1.6.3 Editing .............................................. 61 1.6.3.1 Moving the editing cursor..................... 61 1.6.3.2 Editing Modes ............................... 61 1.6.3.3 Adjusting the Quality Values ................. 64 1.6.3.4 Adjusting the Cutoff Data .................... 64 1.6.3.5 Summary of Editing Commands............... 64 1.6.4 Selections ............................................ 65 1.6.5 Annotations.......................................... 65 1.6.6 Searching ............................................ 67 1.6.6.1 Search by Position ........................... 68 1.6.6.2 Search by Problem ........................... 68 1.6.6.3 Search by Annotation Comments .............. 68 1.6.6.4 Search by Tag Type .......................... 68 1.6.6.5 Search by Sequence .......................... 68 1.6.6.6 Search by Quality ............................ 68 1.6.6.7 Search by Consensus Quality.................. 68 1.6.6.8 Search by file ................................ 69 1.6.6.9 Search by Reading Name ..................... 69 1.6.6.10 Search by Edit.............................. 69 1.6.6.11 Search by Evidence for Edit (1) .............. 69 1.6.6.12 Search by Evidence for Edit (2) .............. 69 1.6.6.13 Search by Discrepancies ..................... 70 1.6.7 The Commands Menu ................................ 70 1.6.7.1 Search ...................................... 70 1.6.7.2 Create Tag .................................. 70 1.6.7.3 Edit Tag .................................... 70 1.6.7.4 Delete Tag .................................. 70 1.6.7.5 Save Contig ................................. 70 1.6.7.6 Dump Contig to File ......................... 70 1.6.7.7 Save Consensus Trace ........................ 71 1.6.7.8 List Confidence .............................. 71 1.6.7.9 Report Mutations ............................ 71 1.6.7.10 Select Primer ............................... 72 1.6.7.11 Align ...................................... 72 1.6.7.12 Shuffle Pads ................................ 72 1.6.7.13 Remove Reading ............................ 72 1.6.7.14 Break Contig ............................... 72 1.6.8 The Settings Menu ................................... 72 1.6.8.1 Status Line .................................. 73 1.6.8.2 Trace Display ................................ 74 1.6.8.3 Consensus Algorithm ......................... 75 1.6.8.4 Highlight Disagreements ...................... 76 1.6.8.5 Compare Strands ............................ 76 1.6.8.6 Toggle auto-save ............................. 76 v 1.6.8.7 3 Character Amino Acids ..................... 76 1.6.8.8 Group Readings by Templates ................ 76 1.6.8.9 Show Reading and Consensus Quality ......... 76 1.6.8.10 Show edits ................................. 77 1.6.8.11 Show Unpadded Positions ................... 77 1.6.8.12 Set Active Tags ............................. 77 1.6.8.13 Set Output List............................. 78 1.6.8.14 Set Default Confidences ..................... 78 1.6.8.15 Set or unset saving of undo .................. 78 1.6.9 Removing Readings ................................... 78 1.6.10 Primer Selection..................................... 79 1.6.10.1 Parameters ................................. 79 1.6.10.2 Template selection .......................... 80 1.6.11 Traces .............................................. 80 1.6.12 Reference Sequence and Traces ....................... 82 1.6.12.1 Reference sequences ......................... 82 1.6.12.2 Reference traces ............................ 83 1.6.13 The Editor Information Line ......................... 83 1.6.13.1 Reading Information ........................ 84 1.6.13.2 Contig Information.......................... 85 1.6.13.3 Tag Information ............................ 85 1.6.13.4 Base Information ........................... 85 1.6.14 The Join Editor ..................................... 86 1.6.15 Using Several Editors at Once ........................ 86 1.6.16 Quitting the Editor .................................. 87 1.6.17 Editing Techniques .................................. 87 1.6.17.1 Consensus and Quality Cutoffs ............... 87 1.6.17.2 Editing
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