Table of Contents How to use this guide ........................................................................................................... 4 1 Introduction to DNA Master ......................................................................................... 6 1.1 DNA Master overview ........................................................................................................................................ 6 1.2 Installation .............................................................................................................................................................. 6 1.3 Quick Start Guide .................................................................................................................................................. 6 1.4 DNA Master program structure ..................................................................................................................... 6 1.5 Analysis programs running within DNA Master ..................................................................................... 7 1.5.1 Glimmer ............................................................................................................................................................................ 7 1.5.2 GeneMark ......................................................................................................................................................................... 8 1.5.3 Aragorn ............................................................................................................................................................................. 8 1.6 Setting Preferences .............................................................................................................................................. 9 1.6.1 Set Default Translation Table .................................................................................................................................. 9 1.6.2 Set color preferences ................................................................................................................................................... 9 1.6.3 Set start codon choices ............................................................................................................................................ 10 1.6.4 Set default values for BLAST searches .............................................................................................................. 11 1.6.5 Choose a default location for saving files ........................................................................................................ 11 1.6.6 Finishing up your Preference settings .............................................................................................................. 12 1.7 Getting help .......................................................................................................................................................... 12 1.8 Checking for updates ....................................................................................................................................... 13 2 Provisional Cluster assignment of your phage ................................................... 15 2.1 Overview ............................................................................................................................................................... 15 2.2 BLASTing your sequence against the mycobacteriophage database .......................................... 15 2.3 Cluster assignment ........................................................................................................................................... 18 3 Importing your phage genome sequence into DNA Master ............................ 21 3.1 Overview ............................................................................................................................................................... 21 3.2 Where do I get my phage genome sequence from? ............................................................................ 21 3.3 Importing your DNA sequence into DNA Master ................................................................................ 22 3.4 Reverse-complementing your sequence ................................................................................................. 24 4 Performing and viewing a rapid automated annotation of your genome . 25 4.1 Overview ............................................................................................................................................................... 25 4.2 Running Auto-Annotate .................................................................................................................................. 25 4.3 Saving your file ................................................................................................................................................... 27 4.4 Looking at the output of your automated annotation ....................................................................... 27 4.4.1 Viewing the documentation .................................................................................................................................. 28 4.4.2 Viewing features in the Feature Table .............................................................................................................. 29 4.4.3 Viewing the sequence in the Sequence tab ..................................................................................................... 31 4.4.4 Viewing ORFs in the Frames window ............................................................................................................... 32 4.5 Running the BLAST function ........................................................................................................................ 35 4.6 Re-opening an archived (saved) file ......................................................................................................... 37 1 5 Gathering additional information for refining your annotation .................. 39 5.1 Generating a six-frame translation ............................................................................................................ 39 5.2 Generating a provisional genome map in DNA Master ..................................................................... 42 5.3 Generating a graphical output from GeneMark ................................................................................... 43 6 Using Phamerator to assist with annotation ....................................................... 47 6.1 Overview ............................................................................................................................................................... 47 6.2 Why Phamerator is useful to you at this stage of your annotation ............................................. 47 6.3 How did my genome get into Phamerator already? ........................................................................... 47 6.4 Making Phamerator maps ............................................................................................................................. 48 6.5 Understanding and using the genome maps made by Phamerator ............................................ 50 6.6 Viewing nucleotide sequence similarities in Phamerator ............................................................... 52 6.7 Other Phamerator features ........................................................................................................................... 54 6.8 Saving Phamerator maps ............................................................................................................................... 55 7 Guiding Principles of Bacteriophage Genome Annotation ............................. 57 7.1 Overview ............................................................................................................................................................... 57 7.2 The Guiding Principles .................................................................................................................................... 57 8 Gene by gene: evaluating and improving your draft annotation ................. 61 8.1 Overview ............................................................................................................................................................... 61 8.2 Button-pushing mechanics reserved for Section 9 ............................................................................. 61 8.3 Decision Tree for evaluating the draft annotation .............................................................................. 61 8.4 Evaluating protein-coding gene calls ........................................................................................................ 63 8.4.1 Is the designation of this ORF as a gene well-supported? ........................................................................ 63 8.4.2 Is the called start site for this gene the best possible choice? ................................................................ 66 8.4.3 Is this gene part of a programmed translational frameshift? ................................................................. 70 8.4.4 Does this gene contain an intron? ...................................................................................................................... 72 8.4.5 Does this gene wrap around the ends of the genome? .............................................................................. 73 8.5 Checking gaps in the draft annotation