Outline for ContigExpress workshop

Transcription

1 Outline for ContigExpress workshop Importing fragments Individual editing Group editing Algorithm Generate contigs and editing contigs 1203 G G T C G A C C C A C G C G T C C G G A A C T T A C T A A A A C C C A C A Fragment PA044D9. XT7 0 G G T C G A C C C A C G C G T C C G G A A C T T A C T A A A A C C C A C A Thomas Randall, PhD tarandal@ .unc.edu

2 ContigExpress is based on the CAP3 assembly algorithm Genome Res. 9: 868

3 ContigExpress Assembly of chromatographs Input: chromatographs Removal of poor end regions of reads Clipping Individual editing Trimming: 5 and 3 Vector polya Quality scores Computation of overlaps between reads Removal of false overlaps Construction of contigs Overlap Opportunity for user input Viewer Contig editing

4 Sequencher vs ContigExpress Different algorithms, assembly options End trimming and vector trimming very similar Sequencher vector trimming more reliable Both use quality scores UNC Sequencing facility does not give quality scores (problems with ABI basecalling algorithm) Integration of ContigExpress into Vector NTI is very convenient, graphics better No need to learn both Most think Sequencher is superior to ContigExpress Assembly to Reference (SNP finding) and capacity for larger sequences Sequencher licensed for UNC-CH only, ContigExpress is licensed by us to free to UNC-CH, NCSU and ECU

5 Importing chromatographs into a ContigExpress project Open a new assembly project under the Assembly drop down menu either in a Vector NTI or Explorer Database window Either drag chromatographs Into a new project or Use the Import function Project > Add Fragments If using the Add Fragments option, set File type to All Files, as this function is sensitive to extensions Project may contain more than one type of file, i.e., text, abi are both allowed in one project

6 Quality scores q = -10 log 10 (p) Phred quality score Probability that the base is called wrong 1 in 10 1 in in 1,000 1 in 10,000 1 in 100,000 Accuracy of the base call 90% 99% 99.9% 99.99% % G G T C G A C C C A C G C G T C C Spacing G G T C G A C C C A C G C G T C C F rag m ent P A D 9. X T 7 Height >PA003A8.XT ABI

7 Forward-Reverse constraints: forces paired end reads to be assembled in opposite primer R orientations INSERT 3 kb ORI primer F insert in puc bp May want seqf and seqr From this clone to be in Opposite orientations Within 2-4 kb of each other P(BLA) APr 2-4 kb seqf seqr contig

8 Never click this option Overlap length cutoff, Overlap % identity cutoff are most important, defaults are good

9 Sequence editing in CE Autoclipping Assembly > Assembly Setup > Clipping set ON as default, can set options, uses quality scores if available Irreversible within a CE project (cant undo) Trim Selected Fragment Ends user defined options Treats 5 and 3 ends of traces differently Reversible can change individual traces back to experimental Trim Selected Fragment Ends for Vector Contamination user defined options Need to have Vector in the Explorer Database Directions on next page, very specific directions needed Reversible can change individual traces back to experimental Individual editing can individually edit chromats in a project Contig editing once contig is built, the consensus can be edited Quick option use autoclipping with contig editing All editing options can be applied within a single project editing applies only to traces within that project

10 Vector Trimming Function Put vector in Database Create reverse complement of vector with new name vector rc For both: Tools>Send to>polylinker to ContigExpress When in the Trim Selected Fragments for Vector Contamination window Highlight both sequences Add restriction enzymes from Enzlist25.dat Highlight cloning site (try site downstream of primer used for sequencing first), then other if different Click OK Calculate DO ALL OF THE ABOVE Also -

11 Use Camera to capture graphics 1019 C G A C A A C T G C T T G C G C C G C T T G C C A A A G N G A N N T Fragment PA014C6. XT7 Click within pane to be captured and then Click on the Camera icon. The ability to capture graphics is limited. Only the window in view can be captured with the camera function for either individual chromatographs and the Alignment Pane of a contig.» PA044D9.XT7(361)» PA047E7.XT7(381)» PA014C6.XT7(381)» PA016D1.XT7(293)» PA017H6.XT7(340)» PA016F11.XT7(374)» PA033F5.XT7(381)» PH047G8.XT7(291)» PA018F1.XT7(381)» PA036F12.XT7(352)» PA044B5.XT7(361)» PA019_C9.T7X(394) 0 C G A C A A C T G C T T G C G C C G C T T G C C A A A G N G A N N T Consensus(394) PA044D9.XT7: 34» PA047E7.XT7: 14» 603 PA014C6.XT7: 14» 575 PA017H6.XT7: 55» 568 PA016F11.XT7: 21» 577 PA016D1.XT7: 102» 582 The entire Graph Pane of a contig can be captured PA033F5.XT7: 14» 582 PH047G8.XT7: 104» 588 PA018F1.XT7: 14» 474 PA036F12.XT7: 43» 531 PA044B5.XT7: 34» 486 PA019_C9.T7X: 1»

12 Some hints Contigs open in read only click on icon E in upper left to change to editing mode Fragment > Revert to Experimental will eliminate editing changes done on single traces Save assembly project initially after opening as the Revert to Saved option is the only way to Undo group editing changes. Save assembly occasionally thereafter Use Edit > Dismiss Assembly to undo assembly Watch for extensions when importing, file does not need to have correct extension to import Can also drag files in; project can contain multiple types of files When a contig is opened and in Editing mode, right click on the name of a sequence to set that sequence as a reference Use autoclipping to avoid trimming options, autoclipping is irreversible within a project unless the unedited project has been previously been saved Importing a chromatograph into a Project is the only means of putting the sequence into the Explorer Database; once in a project simply drag selected chromatographs into the Database. The Consensus of a Contig can also be imported into the Database this way.