CodonCode Aligner User Manual - Sequence Assembly and Alignment

319 Pages·2015·6.82 MB·English

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