Table Of Content

CodonCode Aligner User Manual CodonCode Aligner User Manual Table of Contents About CodonCode Aligner.................................................................................................................................1 System Requirements..............................................................................................................................1 Licenses....................................................................................................................................................1 Licenses for CodonCode Aligner.......................................................................................................................3 Demo Mode.............................................................................................................................................3 Time-limited Trials..................................................................................................................................3 Single-user Licenses................................................................................................................................4 Using Phred and Phrap from CodonCode Aligner............................................................................4 Replacement License Keys...............................................................................................................5 License Server Licenses...........................................................................................................................5 Firewall ports for License Server use................................................................................................6 CodonCode Aligner Features.............................................................................................................................7 Aligner Windows and Views..................................................................................................................7 The Main Window.............................................................................................................................7 Project Window.................................................................................................................................8 Base View Window...........................................................................................................................8 Trace Window..................................................................................................................................9 Quality View Window.....................................................................................................................10 Contig View Window......................................................................................................................10 Restriction Map Window................................................................................................................11 Customizable Toolbars..........................................................................................................................12 Regions of Interest.................................................................................................................................12 Take the Quick Tour!.............................................................................................................................13 Usage Tips for CodonCode Aligner.................................................................................................................14 Quality Values In CodonCode Aligner...........................................................................................................15 Quality values explained........................................................................................................................15 Where quality values come from...........................................................................................................15 Artificial qualities..................................................................................................................................16 Gap qualities..........................................................................................................................................16 Viewing qualities...................................................................................................................................16 Additional Reading................................................................................................................................17 Aligner Projects.................................................................................................................................................18 Working with Aligner Projects........................................................................................................................20 Creating New Projects...........................................................................................................................20 Opening Existing Projects.....................................................................................................................21 Saving Projects......................................................................................................................................21 Closing Projects.....................................................................................................................................21 Adding Sample Files to Aligner Projects........................................................................................................22 Opening Single Sample Files.................................................................................................................22 Adding Several Sample Files.................................................................................................................22 Adding a Subset of Samples..................................................................................................................23 i CodonCode Aligner User Manual Table of Contents Adding Sample Files to Aligner Projects Adding Entire Folders of Sample Files..................................................................................................24 Adding Entire Assemblies.....................................................................................................................24 Importing Sequencher Projects (CAF Files)...................................................................................25 Importing Phrap Assemblies...........................................................................................................26 Tags in Phrap assemblies................................................................................................................26 Import from GenBank............................................................................................................................26 Creating new text sequences..................................................................................................................27 Compatible File Formats.......................................................................................................................28 Sample Names.......................................................................................................................................29 Organizing Samples And Contigs In Folders.................................................................................................31 Creating Folders.....................................................................................................................................31 Moving Samples and Contigs to Folders...............................................................................................31 Renaming Folders..................................................................................................................................31 Deleting Folders.....................................................................................................................................31 Removing Samples from an Aligner Project..................................................................................................33 Base Calling with PHRED................................................................................................................................34 How to call bases with PHRED from Aligner.......................................................................................34 Prerequisites....................................................................................................................................34 What Aligner does...........................................................................................................................35 Editing the PHRED parameter file........................................................................................................35 Base calling problems............................................................................................................................37 Cannot find base calling program...................................................................................................37 Missing entries in the Phred parameter file.....................................................................................38 Problems reading the Phred parameter file.....................................................................................39 .W....r.o..n..g.. .c..o..m...m...a..n..d.. .l.i.n..e.. .p..a..r.a..m...e..t.e..r.s........................................................................................................................................................................................................3399 Problem running the workstation version of Phred.........................................................................40 More about the Phred parameter file.....................................................................................................40 About Phred...........................................................................................................................................41 End Clipping......................................................................................................................................................43 End Clipping Parameters.......................................................................................................................44 End Clipping Algorithms.......................................................................................................................45 Method 1: Maximizing regions with error rates below a given threshold......................................45 Method 2: Using separate criteria at the start and the end of the sequence....................................45 Trimming Vector Sequences............................................................................................................................47 Vector Library Files.........................................................................................................................................49 Using UniVec Library Files...................................................................................................................49 Using Custom Vector Files....................................................................................................................49 Assembly and Alignment..................................................................................................................................51 Sequence Assembly...............................................................................................................................51 NGS Assembly......................................................................................................................................51 ii CodonCode Aligner User Manual Table of Contents Assembly and Alignment Alignment with Clustal, Muscle, or based on protein translations (with MACSE)..............................52 Alignment to a Reference Sequence......................................................................................................52 Alignment with Bowtie2........................................................................................................................53 Sequence Assembly...........................................................................................................................................54 Before assembling..................................................................................................................................54 How to assemble....................................................................................................................................54 Advanced assembly options...................................................................................................................54 Assemble with preprocessing................................................................................................................55 Assemble in groups................................................................................................................................56 Assemble in groups by name part...................................................................................................57 Assemble in groups by multiplex sequence tag.............................................................................59 Compare contigs to each other...............................................................................................................60 Assemble from scratch...........................................................................................................................63 Sequence Assembly With Phrap......................................................................................................................64 How Aligner assembles using Phrap.....................................................................................................64 Things to Note for Phrap Assemblies....................................................................................................64 About Phrap...........................................................................................................................................65 Alignments to a Reference Sequence...............................................................................................................66 Adding Samples to Alignments.............................................................................................................67 Advanced Alignments............................................................................................................................67 Align with Preprocessing.......................................................................................................................68 Align to Reference in Groups................................................................................................................69 Align to Reference from Scratch..........................................................................................................70 Contigs...............................................................................................................................................................71 Unassembling Contigs......................................................................................................................................72 Aligner Algorithms for Assembly and Alignments........................................................................................73 Alignment to a Reference Sequence......................................................................................................73 Assembly...............................................................................................................................................73 Consensus Calculation...........................................................................................................................74 Quality-based Consensus................................................................................................................74 Majority Consensus.........................................................................................................................75 Inclusive Consensus........................................................................................................................76 Percentage-based Consensus...........................................................................................................76 Using the Reference Sequence as Consensus..................................................................................76 Rebuilding the Consensus Sequence...............................................................................................77 Local, large gap, and end-to-end alignments.........................................................................................77 NGS Data Analysis and Assembly...................................................................................................................79 Metagenomics........................................................................................................................................79 Pre-processing tools...............................................................................................................................79 NGS Assembly......................................................................................................................................79 iii CodonCode Aligner User Manual Table of Contents NGS Data Analysis and Assembly NGS Alignments....................................................................................................................................79 Cluster Sequences.............................................................................................................................................80 How Aligner Clusters............................................................................................................................81 Clustering Result Files...........................................................................................................................81 Advanced Clustering Options................................................................................................................81 L...i.m...i.t.a..t.i.o..n..s...........................................................................................................................................................................................................................................................................................8823 Identify Sequences............................................................................................................................................84 How to Identify Sequences....................................................................................................................84 Query Sequences.............................................................................................................................84 Reference Sequences.......................................................................................................................84 Stringency and Options...................................................................................................................85 Identify Sequences Results....................................................................................................................87 Identify Sequences Algorithm...............................................................................................................87 Limitations.............................................................................................................................................88 NGS Preprocessing Tools.................................................................................................................................89 Trimming Reads and Removing Adapter Sequences............................................................................89 Error Correction with SparseAssembler................................................................................................92 Other NGS Tools...................................................................................................................................93 NGS Assembly...................................................................................................................................................94 NGS Assembly with CodonCode Aligner.............................................................................................94 NGS Assembly with Tadpole................................................................................................................95 NGS Assembly with SparseAssembler..................................................................................................96 Other NGS Assembly Programs............................................................................................................97 Scaffolding.............................................................................................................................................97 Alignments with Bowtie 2.................................................................................................................................98 Viewing Bowtie2 alignments with Tablet...........................................................................................102 Regions of Interest: Features.........................................................................................................................104 Defining Features.................................................................................................................................104 Moving to Features..............................................................................................................................105 View, Print, Export Features................................................................................................................106 Finding Mutations...........................................................................................................................................107 Prerequisites.........................................................................................................................................107 How To Find SNPs..............................................................................................................................107 Fixing Errors........................................................................................................................................110 Tags for Finding Mutations.................................................................................................................111 Defining the Coding Region..........................................................................................................111 Numbering the Coding Sequence..................................................................................................113 Excluding Regions from Analysis.................................................................................................115 How Aligner Finds SNPs.....................................................................................................................116 Limitations...........................................................................................................................................116 iv CodonCode Aligner User Manual Table of Contents Heterozygous Insertions and Deletions.........................................................................................................118 Finding Heterozygous Indels...............................................................................................................119 Hetero Indel Scores.......................................................................................................................120 False Negatives..............................................................................................................................120 False Positives...............................................................................................................................121 Splitting Heterozygous Indels..............................................................................................................121 Processing Heterozygous Indels..........................................................................................................121 Limitations...........................................................................................................................................124 Processing Pre-Requisites and Limitations...................................................................................124 Interpreting Results.......................................................................................................................124 Acknowledgements.......................................................................................................................125 Methylation Analysis......................................................................................................................................126 Prerequisites.........................................................................................................................................126 Raw ABI Data...............................................................................................................................126 Reference Sequence Alignments...................................................................................................126 How to Analyze Methylation...............................................................................................................126 Viewing Raw Trace Data.....................................................................................................................127 Stretching raw data..............................................................................................................................128 Methylation Analysis Algorithm.........................................................................................................128 Primer Design..................................................................................................................................................130 How to Pick Primers............................................................................................................................130 Primer Design Parameters....................................................................................................................130 PCR Parameters.............................................................................................................................131 Sequencing Parameters..................................................................................................................132 Conditions & Advanced Settings Parameters...............................................................................132 Primer Results......................................................................................................................................134 Exporting Primers................................................................................................................................136 Additional Information........................................................................................................................137 RFLP Analysis.................................................................................................................................................138 Prerequisites.........................................................................................................................................138 How to Analyze RFLPs.......................................................................................................................138 Samples with Different Lengths....................................................................................................138 Analysis Options..................................................................................................................................138 Analysis Results...................................................................................................................................140 Dot Plots...........................................................................................................................................................142 Editing Samples...............................................................................................................................................146 Windows for Editing Samples........................................................................................................................149 Cursor Positioning and Movement................................................................................................................150 Moving to Features, Ambiguities, Mismatches or Edited Bases.........................................................150 v CodonCode Aligner User Manual Table of Contents Setting Base Numbers.....................................................................................................................................151 Selecting Bases.................................................................................................................................................152 Selecting from sequence start to current cursor position.....................................................................152 Selecting from current cursor position to the end of a sequence.........................................................152 Selecting all bases in a sequence.........................................................................................................152 Selecting Ranges of Bases...................................................................................................................152 Selecting Samples and Contigs......................................................................................................................154 Changing Bases...............................................................................................................................................155 Manual Editing....................................................................................................................................155 Making Bases Lower or Upper Case...................................................................................................155 Automatic Edits...................................................................................................................................155 Match Consensus...........................................................................................................................156 Call Second Peaks Higher Than....................................................................................................156 Change Ambiguities to Single Bases............................................................................................157 Change Low Quality Bases to N...................................................................................................157 Undo Auto Edits............................................................................................................................157 Change Bases Options...................................................................................................................157 Deleting Bases..................................................................................................................................................159 Using the Keyboard to Delete Bases...................................................................................................159 Menus for Deleting Bases....................................................................................................................159 Deleting Samples.............................................................................................................................................160 Moving Gaps and Samples.............................................................................................................................161 Moving gaps in contigs........................................................................................................................161 Moving samples in contigs..................................................................................................................161 Moving samples to "Unassembled Samples"......................................................................................161 Inserting Gaps and Bases...............................................................................................................................162 Adding Bases at the End of Reads.......................................................................................................162 Reverse Complementing.................................................................................................................................163 Editing Sample Information..........................................................................................................................164 Changing Sample Names.....................................................................................................................164 Viewing Chromatogram Information..................................................................................................164 Viewing Sample Tags..........................................................................................................................166 Confirming Tags............................................................................................................................167 Viewing "Local" Tags...................................................................................................................167 Adding Tags.........................................................................................................................................168 A...d..d..i.n..g.. .T...a..g..s. .t.o.. .A...l.l. .S...e.q..u..e..n..c..e..s..........................................................................................................................................................................................................................116689 vi CodonCode Aligner User Manual Table of Contents Saving Edits.....................................................................................................................................................170 Undo and Redo................................................................................................................................................171 Copy and Paste................................................................................................................................................172 Copy (selected sequence).....................................................................................................................172 Paste.....................................................................................................................................................172 Editing Contigs................................................................................................................................................173 Adding Samples to Contigs............................................................................................................................174 Duplicating samples.............................................................................................................................174 Merging Contigs..............................................................................................................................................175 Removing Samples from Contigs..................................................................................................................176 Deleting Parts of Contigs................................................................................................................................177 Removing Consensus Gaps............................................................................................................................178 How to remove consensus gaps...........................................................................................................178 Alignment Locations - Start and End...........................................................................................................180 Reverse Complementing.................................................................................................................................181 Splitting Contigs..............................................................................................................................................182 Unassembling Contigs....................................................................................................................................184 Roundtrip Editing...........................................................................................................................................185 How To Roundtrip Edit with CodonCode Aligner..............................................................................185 Limitations...........................................................................................................................................186 Editing Contig Information...........................................................................................................................187 Search for Sequences......................................................................................................................................188 BLAST Searches.............................................................................................................................................189 MegaBLAST........................................................................................................................................189 Nucleotide (blastn)...............................................................................................................................189 Translated (blastx)...............................................................................................................................189 Translated (tblastx)..............................................................................................................................189 Exporting.........................................................................................................................................................190 vii CodonCode Aligner User Manual Table of Contents Export Project Summary...............................................................................................................................191 Exporting Samples..........................................................................................................................................193 Export options for FASTA files...........................................................................................................194 Exporting SCF files.............................................................................................................................195 Exporting Consensus Sequences....................................................................................................................196 E...x..p..o..r.t. .o..p..t.i..o..n..s. .f.o..r.. .F..A...S..T...A.. .f..i.l.e..s......................................................................................................................................................................................................................119968 Exporting Assemblies.....................................................................................................................................198 ACE Format Exports...........................................................................................................................198 NEXUS/PAUP Format Exports...........................................................................................................199 Phylip Format Exports.........................................................................................................................199 Other Formats for Exporting Assemblies............................................................................................200 Exporting Features.........................................................................................................................................201 Exporting Aligner Projects in the Old Format............................................................................................203 Exporting Protein Translation.......................................................................................................................204 Exporting Differences.....................................................................................................................................205 Exporting Trees...............................................................................................................................................207 Aligner Windows.............................................................................................................................................208 The Project View Window...................................................................................................................208 Selecting Samples and Contigs.....................................................................................................208 Menu shortcuts: Buttons and Popup menus..................................................................................209 Project View Columns And Sorting..............................................................................................209 Status messages.............................................................................................................................210 Colored Labels..............................................................................................................................210 Trace View.......................................................................................................................................................212 General Information.............................................................................................................................212 Opening a Trace View.........................................................................................................................212 Scrolling and Scaling in Trace View...................................................................................................212 Trace Sharpening.................................................................................................................................213 Colors and Highlighting.......................................................................................................................214 Automatic Trace Selection...................................................................................................................214 Hiding Some Traces.............................................................................................................................214 Printing Traces.....................................................................................................................................216 Base View Window.........................................................................................................................................217 Quality View Window.....................................................................................................................................219 viii CodonCode Aligner User Manual Table of Contents Contig View Window......................................................................................................................................220 Moving Around....................................................................................................................................221 Contig Overview Panel........................................................................................................................222 Coverage graph in the overview panel..........................................................................................222 Navigating using the overview panel............................................................................................222 Changing the display of the sample arrows...................................................................................223 Contig Difference Table......................................................................................................................224 Display options for the difference table........................................................................................225 Navigating using the difference table............................................................................................228 Aligned Bases and Consensus Protein Translation..............................................................................228 Phylogenetic Trees...............................................................................................................................229 Split Contig From Tree..................................................................................................................232 Building Trees for Selected Bases Only........................................................................................233 Masking Bases Matching the Consensus.............................................................................................234 Zooming in the Contig View...............................................................................................................235 Sorting Reads in the Contig View.......................................................................................................237 Automatic Trace Selection...................................................................................................................238 Printing Contigs...................................................................................................................................238 Feature Window..............................................................................................................................................240 Restriction Map View.....................................................................................................................................242 Single Line Map...................................................................................................................................242 Multi Line Map....................................................................................................................................242 Text Map..............................................................................................................................................243 Virtual Gel...........................................................................................................................................244 Selecting Fragments.............................................................................................................................245 Restriction Enzymes in Aligner...........................................................................................................247 Closing Windows.............................................................................................................................................248 Scripting CodonCode Aligner........................................................................................................................249 Preferences and Settings.................................................................................................................................253 Alignment Preferences....................................................................................................................................255 Algorithm.............................................................................................................................................256 Minimum Percent Identity...................................................................................................................256 Minimum Overlap Length...................................................................................................................256 Minimum Alignment Score.................................................................................................................256 Maximum Unaligned End Overlap......................................................................................................257 Bandwidth (Maximum Gap Size)........................................................................................................257 Word Length........................................................................................................................................258 Match scoring.......................................................................................................................................258 Clipping Uncovered Regions...............................................................................................................258 Reverse-complement for external programs........................................................................................259 Restoring default parameters...............................................................................................................259 ix

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CodonCode Aligner User Manual - Sequence Assembly and Alignment PDF

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CodonCode Aligner User Manual CodonCode Aligner User Manual Table of Contents About CodonCode Aligner.................................................................................................................................1 System Requirements..............................................................................................................................1 Licenses....................................................................................................................................................1 Licenses for CodonCode Aligner.......................................................................................................................3 Demo Mode.............................................................................................................................................3 Time-limited Trials..................................................................................................................................3 Single-user Licenses................................................................................................................................4 Using Phred and Phrap from CodonCode Aligner............................................................................4 Replacement License Keys...............................................................................................................5 License Server Licenses...........................................................................................................................5 Firewall ports for License Server use................................................................................................6 CodonCode Aligner Features.............................................................................................................................7 Aligner Windows and Views..................................................................................................................7 The Main Window.............................................................................................................................7 Project Window.................................................................................................................................8 Base View Window...........................................................................................................................8 Trace Window..................................................................................................................................9 Quality View Window.....................................................................................................................10 Contig View Window......................................................................................................................10 Restriction Map Window................................................................................................................11 Customizable Toolbars..........................................................................................................................12 Regions of Interest.................................................................................................................................12 Take the Quick Tour!.............................................................................................................................13 Usage Tips for CodonCode Aligner.................................................................................................................14 Quality Values In CodonCode Aligner...........................................................................................................15 Quality values explained........................................................................................................................15 Where quality values come from...........................................................................................................15 Artificial qualities..................................................................................................................................16 Gap qualities..........................................................................................................................................16 Viewing qualities...................................................................................................................................16 Additional Reading................................................................................................................................17 Aligner Projects.................................................................................................................................................18 Working with Aligner Projects........................................................................................................................20 Creating New Projects...........................................................................................................................20 Opening Existing Projects.....................................................................................................................21 Saving Projects......................................................................................................................................21 Closing Projects.....................................................................................................................................21 Adding Sample Files to Aligner Projects........................................................................................................22 Opening Single Sample Files.................................................................................................................22 Adding Several Sample Files.................................................................................................................22 Adding a Subset of Samples..................................................................................................................23 i CodonCode Aligner User Manual Table of Contents Adding Sample Files to Aligner Projects Adding Entire Folders of Sample Files..................................................................................................24 Adding Entire Assemblies.....................................................................................................................24 Importing Sequencher Projects (CAF Files)...................................................................................25 Importing Phrap Assemblies...........................................................................................................26 Tags in Phrap assemblies................................................................................................................26 Import from GenBank............................................................................................................................26 Creating new text sequences..................................................................................................................27 Compatible File Formats.......................................................................................................................28 Sample Names.......................................................................................................................................29 Organizing Samples And Contigs In Folders.................................................................................................31 Creating Folders.....................................................................................................................................31 Moving Samples and Contigs to Folders...............................................................................................31 Renaming Folders..................................................................................................................................31 Deleting Folders.....................................................................................................................................31 Removing Samples from an Aligner Project..................................................................................................33 Base Calling with PHRED................................................................................................................................34 How to call bases with PHRED from Aligner.......................................................................................34 Prerequisites....................................................................................................................................34 What Aligner does...........................................................................................................................35 Editing the PHRED parameter file........................................................................................................35 Base calling problems............................................................................................................................37 Cannot find base calling program...................................................................................................37 Missing entries in the Phred parameter file.....................................................................................38 Problems reading the Phred parameter file.....................................................................................39 .W....r.o..n..g.. .c..o..m...m...a..n..d.. .l.i.n..e.. .p..a..r.a..m...e..t.e..r.s........................................................................................................................................................................................................3399 Problem running the workstation version of Phred.........................................................................40 More about the Phred parameter file.....................................................................................................40 About Phred...........................................................................................................................................41 End Clipping......................................................................................................................................................43 End Clipping Parameters.......................................................................................................................44 End Clipping Algorithms.......................................................................................................................45 Method 1: Maximizing regions with error rates below a given threshold......................................45 Method 2: Using separate criteria at the start and the end of the sequence....................................45 Trimming Vector Sequences............................................................................................................................47 Vector Library Files.........................................................................................................................................49 Using UniVec Library Files...................................................................................................................49 Using Custom Vector Files....................................................................................................................49 Assembly and Alignment..................................................................................................................................51 Sequence Assembly...............................................................................................................................51 NGS Assembly......................................................................................................................................51 ii CodonCode Aligner User Manual Table of Contents Assembly and Alignment Alignment with Clustal, Muscle, or based on protein translations (with MACSE)..............................52 Alignment to a Reference Sequence......................................................................................................52 Alignment with Bowtie2........................................................................................................................53 Sequence Assembly...........................................................................................................................................54 Before assembling..................................................................................................................................54 How to assemble....................................................................................................................................54 Advanced assembly options...................................................................................................................54 Assemble with preprocessing................................................................................................................55 Assemble in groups................................................................................................................................56 Assemble in groups by name part...................................................................................................57 Assemble in groups by multiplex sequence tag.............................................................................59 Compare contigs to each other...............................................................................................................60 Assemble from scratch...........................................................................................................................63 Sequence Assembly With Phrap......................................................................................................................64 How Aligner assembles using Phrap.....................................................................................................64 Things to Note for Phrap Assemblies....................................................................................................64 About Phrap...........................................................................................................................................65 Alignments to a Reference Sequence...............................................................................................................66 Adding Samples to Alignments.............................................................................................................67 Advanced Alignments............................................................................................................................67 Align with Preprocessing.......................................................................................................................68 Align to Reference in Groups................................................................................................................69 Align to Reference from Scratch..........................................................................................................70 Contigs...............................................................................................................................................................71 Unassembling Contigs......................................................................................................................................72 Aligner Algorithms for Assembly and Alignments........................................................................................73 Alignment to a Reference Sequence......................................................................................................73 Assembly...............................................................................................................................................73 Consensus Calculation...........................................................................................................................74 Quality-based Consensus................................................................................................................74 Majority Consensus.........................................................................................................................75 Inclusive Consensus........................................................................................................................76 Percentage-based Consensus...........................................................................................................76 Using the Reference Sequence as Consensus..................................................................................76 Rebuilding the Consensus Sequence...............................................................................................77 Local, large gap, and end-to-end alignments.........................................................................................77 NGS Data Analysis and Assembly...................................................................................................................79 Metagenomics........................................................................................................................................79 Pre-processing tools...............................................................................................................................79 NGS Assembly......................................................................................................................................79 iii CodonCode Aligner User Manual Table of Contents NGS Data Analysis and Assembly NGS Alignments....................................................................................................................................79 Cluster Sequences.............................................................................................................................................80 How Aligner Clusters............................................................................................................................81 Clustering Result Files...........................................................................................................................81 Advanced Clustering Options................................................................................................................81 L...i.m...i.t.a..t.i.o..n..s...........................................................................................................................................................................................................................................................................................8823 Identify Sequences............................................................................................................................................84 How to Identify Sequences....................................................................................................................84 Query Sequences.............................................................................................................................84 Reference Sequences.......................................................................................................................84 Stringency and Options...................................................................................................................85 Identify Sequences Results....................................................................................................................87 Identify Sequences Algorithm...............................................................................................................87 Limitations.............................................................................................................................................88 NGS Preprocessing Tools.................................................................................................................................89 Trimming Reads and Removing Adapter Sequences............................................................................89 Error Correction with SparseAssembler................................................................................................92 Other NGS Tools...................................................................................................................................93 NGS Assembly...................................................................................................................................................94 NGS Assembly with CodonCode Aligner.............................................................................................94 NGS Assembly with Tadpole................................................................................................................95 NGS Assembly with SparseAssembler..................................................................................................96 Other NGS Assembly Programs............................................................................................................97 Scaffolding.............................................................................................................................................97 Alignments with Bowtie 2.................................................................................................................................98 Viewing Bowtie2 alignments with Tablet...........................................................................................102 Regions of Interest: Features.........................................................................................................................104 Defining Features.................................................................................................................................104 Moving to Features..............................................................................................................................105 View, Print, Export Features................................................................................................................106 Finding Mutations...........................................................................................................................................107 Prerequisites.........................................................................................................................................107 How To Find SNPs..............................................................................................................................107 Fixing Errors........................................................................................................................................110 Tags for Finding Mutations.................................................................................................................111 Defining the Coding Region..........................................................................................................111 Numbering the Coding Sequence..................................................................................................113 Excluding Regions from Analysis.................................................................................................115 How Aligner Finds SNPs.....................................................................................................................116 Limitations...........................................................................................................................................116 iv CodonCode Aligner User Manual Table of Contents Heterozygous Insertions and Deletions.........................................................................................................118 Finding Heterozygous Indels...............................................................................................................119 Hetero Indel Scores.......................................................................................................................120 False Negatives..............................................................................................................................120 False Positives...............................................................................................................................121 Splitting Heterozygous Indels..............................................................................................................121 Processing Heterozygous Indels..........................................................................................................121 Limitations...........................................................................................................................................124 Processing Pre-Requisites and Limitations...................................................................................124 Interpreting Results.......................................................................................................................124 Acknowledgements.......................................................................................................................125 Methylation Analysis......................................................................................................................................126 Prerequisites.........................................................................................................................................126 Raw ABI Data...............................................................................................................................126 Reference Sequence Alignments...................................................................................................126 How to Analyze Methylation...............................................................................................................126 Viewing Raw Trace Data.....................................................................................................................127 Stretching raw data..............................................................................................................................128 Methylation Analysis Algorithm.........................................................................................................128 Primer Design..................................................................................................................................................130 How to Pick Primers............................................................................................................................130 Primer Design Parameters....................................................................................................................130 PCR Parameters.............................................................................................................................131 Sequencing Parameters..................................................................................................................132 Conditions & Advanced Settings Parameters...............................................................................132 Primer Results......................................................................................................................................134 Exporting Primers................................................................................................................................136 Additional Information........................................................................................................................137 RFLP Analysis.................................................................................................................................................138 Prerequisites.........................................................................................................................................138 How to Analyze RFLPs.......................................................................................................................138 Samples with Different Lengths....................................................................................................138 Analysis Options..................................................................................................................................138 Analysis Results...................................................................................................................................140 Dot Plots...........................................................................................................................................................142 Editing Samples...............................................................................................................................................146 Windows for Editing Samples........................................................................................................................149 Cursor Positioning and Movement................................................................................................................150 Moving to Features, Ambiguities, Mismatches or Edited Bases.........................................................150 v CodonCode Aligner User Manual Table of Contents Setting Base Numbers.....................................................................................................................................151 Selecting Bases.................................................................................................................................................152 Selecting from sequence start to current cursor position.....................................................................152 Selecting from current cursor position to the end of a sequence.........................................................152 Selecting all bases in a sequence.........................................................................................................152 Selecting Ranges of Bases...................................................................................................................152 Selecting Samples and Contigs......................................................................................................................154 Changing Bases...............................................................................................................................................155 Manual Editing....................................................................................................................................155 Making Bases Lower or Upper Case...................................................................................................155 Automatic Edits...................................................................................................................................155 Match Consensus...........................................................................................................................156 Call Second Peaks Higher Than....................................................................................................156 Change Ambiguities to Single Bases............................................................................................157 Change Low Quality Bases to N...................................................................................................157 Undo Auto Edits............................................................................................................................157 Change Bases Options...................................................................................................................157 Deleting Bases..................................................................................................................................................159 Using the Keyboard to Delete Bases...................................................................................................159 Menus for Deleting Bases....................................................................................................................159 Deleting Samples.............................................................................................................................................160 Moving Gaps and Samples.............................................................................................................................161 Moving gaps in contigs........................................................................................................................161 Moving samples in contigs..................................................................................................................161 Moving samples to "Unassembled Samples"......................................................................................161 Inserting Gaps and Bases...............................................................................................................................162 Adding Bases at the End of Reads.......................................................................................................162 Reverse Complementing.................................................................................................................................163 Editing Sample Information..........................................................................................................................164 Changing Sample Names.....................................................................................................................164 Viewing Chromatogram Information..................................................................................................164 Viewing Sample Tags..........................................................................................................................166 Confirming Tags............................................................................................................................167 Viewing "Local" Tags...................................................................................................................167 Adding Tags.........................................................................................................................................168 A...d..d..i.n..g.. .T...a..g..s. .t.o.. .A...l.l. .S...e.q..u..e..n..c..e..s..........................................................................................................................................................................................................................116689 vi CodonCode Aligner User Manual Table of Contents Saving Edits.....................................................................................................................................................170 Undo and Redo................................................................................................................................................171 Copy and Paste................................................................................................................................................172 Copy (selected sequence).....................................................................................................................172 Paste.....................................................................................................................................................172 Editing Contigs................................................................................................................................................173 Adding Samples to Contigs............................................................................................................................174 Duplicating samples.............................................................................................................................174 Merging Contigs..............................................................................................................................................175 Removing Samples from Contigs..................................................................................................................176 Deleting Parts of Contigs................................................................................................................................177 Removing Consensus Gaps............................................................................................................................178 How to remove consensus gaps...........................................................................................................178 Alignment Locations - Start and End...........................................................................................................180 Reverse Complementing.................................................................................................................................181 Splitting Contigs..............................................................................................................................................182 Unassembling Contigs....................................................................................................................................184 Roundtrip Editing...........................................................................................................................................185 How To Roundtrip Edit with CodonCode Aligner..............................................................................185 Limitations...........................................................................................................................................186 Editing Contig Information...........................................................................................................................187 Search for Sequences......................................................................................................................................188 BLAST Searches.............................................................................................................................................189 MegaBLAST........................................................................................................................................189 Nucleotide (blastn)...............................................................................................................................189 Translated (blastx)...............................................................................................................................189 Translated (tblastx)..............................................................................................................................189 Exporting.........................................................................................................................................................190 vii CodonCode Aligner User Manual Table of Contents Export Project Summary...............................................................................................................................191 Exporting Samples..........................................................................................................................................193 Export options for FASTA files...........................................................................................................194 Exporting SCF files.............................................................................................................................195 Exporting Consensus Sequences....................................................................................................................196 E...x..p..o..r.t. .o..p..t.i..o..n..s. .f.o..r.. .F..A...S..T...A.. .f..i.l.e..s......................................................................................................................................................................................................................119968 Exporting Assemblies.....................................................................................................................................198 ACE Format Exports...........................................................................................................................198 NEXUS/PAUP Format Exports...........................................................................................................199 Phylip Format Exports.........................................................................................................................199 Other Formats for Exporting Assemblies............................................................................................200 Exporting Features.........................................................................................................................................201 Exporting Aligner Projects in the Old Format............................................................................................203 Exporting Protein Translation.......................................................................................................................204 Exporting Differences.....................................................................................................................................205 Exporting Trees...............................................................................................................................................207 Aligner Windows.............................................................................................................................................208 The Project View Window...................................................................................................................208 Selecting Samples and Contigs.....................................................................................................208 Menu shortcuts: Buttons and Popup menus..................................................................................209 Project View Columns And Sorting..............................................................................................209 Status messages.............................................................................................................................210 Colored Labels..............................................................................................................................210 Trace View.......................................................................................................................................................212 General Information.............................................................................................................................212 Opening a Trace View.........................................................................................................................212 Scrolling and Scaling in Trace View...................................................................................................212 Trace Sharpening.................................................................................................................................213 Colors and Highlighting.......................................................................................................................214 Automatic Trace Selection...................................................................................................................214 Hiding Some Traces.............................................................................................................................214 Printing Traces.....................................................................................................................................216 Base View Window.........................................................................................................................................217 Quality View Window.....................................................................................................................................219 viii CodonCode Aligner User Manual Table of Contents Contig View Window......................................................................................................................................220 Moving Around....................................................................................................................................221 Contig Overview Panel........................................................................................................................222 Coverage graph in the overview panel..........................................................................................222 Navigating using the overview panel............................................................................................222 Changing the display of the sample arrows...................................................................................223 Contig Difference Table......................................................................................................................224 Display options for the difference table........................................................................................225 Navigating using the difference table............................................................................................228 Aligned Bases and Consensus Protein Translation..............................................................................228 Phylogenetic Trees...............................................................................................................................229 Split Contig From Tree..................................................................................................................232 Building Trees for Selected Bases Only........................................................................................233 Masking Bases Matching the Consensus.............................................................................................234 Zooming in the Contig View...............................................................................................................235 Sorting Reads in the Contig View.......................................................................................................237 Automatic Trace Selection...................................................................................................................238 Printing Contigs...................................................................................................................................238 Feature Window..............................................................................................................................................240 Restriction Map View.....................................................................................................................................242 Single Line Map...................................................................................................................................242 Multi Line Map....................................................................................................................................242 Text Map..............................................................................................................................................243 Virtual Gel...........................................................................................................................................244 Selecting Fragments.............................................................................................................................245 Restriction Enzymes in Aligner...........................................................................................................247 Closing Windows.............................................................................................................................................248 Scripting CodonCode Aligner........................................................................................................................249 Preferences and Settings.................................................................................................................................253 Alignment Preferences....................................................................................................................................255 Algorithm.............................................................................................................................................256 Minimum Percent Identity...................................................................................................................256 Minimum Overlap Length...................................................................................................................256 Minimum Alignment Score.................................................................................................................256 Maximum Unaligned End Overlap......................................................................................................257 Bandwidth (Maximum Gap Size)........................................................................................................257 Word Length........................................................................................................................................258 Match scoring.......................................................................................................................................258 Clipping Uncovered Regions...............................................................................................................258 Reverse-complement for external programs........................................................................................259 Restoring default parameters...............................................................................................................259 ix

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CodonCode Aligner Release Notes..275 Checking for Aligner Updates
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