Introduction to Sequencher Tom Randall Center for Bioinformatics tarandal@email.unc.edu
Introduction Importing, viewing and manipulating chromatographs Trimming chromatographs Assembly into contigs Editing and exporting contigs
http://www.genecodes.com/sequencher/tutorials.html pdfs of above are on your computer if Sequencher is installed: C > Program Files > Gene Codes > Sequencher 4.x > Tutorials
http://bioinformatics.unc.edu/software/sequencher/index.htm Licensed to UNC CH only, need an onyen 14 concurrent PC licenses, 12 concurrent Mac licenses For off-campus use VPN Client needs to be installed https://shareware.unc.edu/software.html
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 Sequencher licensed for UNC-CH only, ContigExpress is free to academic/govt users
Sequencher Features Open, view and save chromatogram data into fasta format text file Automatic trimming for quality and for vector/primer Create assemblies for shotgun or EST sequencing project Editing contigs while viewing all relevant trace data Assembling multiple sequences to a user-defined Reference Sequence Detecting and annotating polymorphisms in variance table Displaying restriction maps, ORF maps, protein translation Aligning cdnas to their genomic sequence using the Large Gap algorithm
Manipulating individual chromatographs Cut Map displays RE sites (Select Enzymes to choose) Ruler change sequence display, show translations Sequence > Edit Features in drop down menu adding features to your sequence Sequence > Revert to Experimental change editing of a selected sequence(s) View dropdown menu, change look of sequence
Vector Trimming procedure See handout for adding a vector to the VecBase.txt file (email me also) Vector has to be in VecBase.txt file (C > Program Files > Gene Codes > Sequencher 4.x > VecBase.txt Contains 140 vectors, last updated 1987
Vector Trimming, cont. Select sequences to trim Go to Sequence > Trim Vector Select Choose Insertion Site Now Choose Vector and Select Choose Insertion Site Close Insertion Site Window Go to Sequence > Trim Vector to finish If you want to do a different screen you need to close the application and repeat the process
http://www.ncbi.nlm.nih.gov/vecscreen/vecscreen.html UniVec Database Statistics UniVec build #4.0 (Feb. 20, 2007) Number of sequences represented 1096 (4,332,307 bases)
Assembly options Assemble Automatically no manual decisions except setting parameters Assemble Interactively Assemble one by one based on current parameters Assemble to Reference for resequencing projects, SNP or mutation identification Mindlessly Join assemble irregardless of similiarity Assembly parameters overlap and % identity are most important Assemble with gaps for assembly of cdna and genomic, alternative splicing Consensus view Inclusively uses IUPAC ambiguities, less informative Plurality majority rule ASSEMBLY WITH REALIGNER Realigner is an optional step that can augment the standard assembly algorithm. The ReAligner function evaluates the placement of gaps within a contig and optimizes their placement. ReAligner facilitates editing in the consensus sequence and more clearly displays the effect of insertions and deletions.
SNP Discovery Variance Table for SNP Discovery Example project: C > Program Files > Gene Codes > Sequencher 4.x > Sample Data > SNP Hunting use Assemble to Reference option and Sequence > Compare Bases To > Reference Sequence