The Pregap will preprocess the files and display the chromatogram. The parts of the sequence marked for quality clipping will be highlighted. You can view and check your chromatograms for the following problems.
How clean is your sequence? Are the peaks evenly-spaced and is there baseline 'noise'?
Low baseline noise.
Higher baseline noise
High baseline noise
Mis-spaced peaks or double-peaks
Loss of resolution
Homopolymeric stretches
Select Next file button to view the second chromatogram. When you are done exit the Trev program to signal to the Pregap to continue.
When the pregap finishes processing and assembling sequences the Textual Output will display *** Processing finished *** message.
Exit the Pregap
To view the assembled contig file launch the Gap4 program from the Start Menu.
Open the generated database by selecting File -> Open menu.
Browse to the directory you created and select the file nameofdatabase.0.aux
Right click on the graphical preview of the contig sequence in the Contig Selector window and select Edit contig.
The contig editor will display three sequences: two sequences obtained from chromatograms and the contig sequence. To see the chromatogram for a particular position double click the nucleotide.
Explore the different menus and display options.
Edit the contig sequence if required.
When you are done select File Save consensus form the GAP window.
If you require to save portion of your contig file select single from the Save consensus window. Modify the size of the contig sequence, add “.txt” extension to the name of the contig file to make it more convenient to open and save. The sequence file will be saved in FASTA format. A sequence in FASTA format begins with a greater-than symbol (>), followed by a single-line description and then (starting a new line) lines of sequence data.
2.Identify the sequence using BLAST search (Staden).
The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.
-To launch internet browser and follow to the NCBI blast program web interface follow this link http://www.ncbi.nlm.nih.gov/BLAST/
-Select the Nucleotide-nucleotide BLAST (blastn) link to do nucleotide search. When doing an amino acid query choose blastp.
-Copy and paste your contig sequence into the Search box.
-Options include a number of different sequence databases that can be searched using blastn. The default database is nr, which is the collection of all unique sequences. It contains all non redundant Genbank CDS translations PDB SwissProt PIR PRF entries. The nr database is a good choice for a comprehensive search. More information on the BLAST databases is available at http://www.ncbi.nlm.nih.gov/blast/blast_databases.shtml.
-After you submit your query, you will be taken to the formatting BLAST page.
-The formatting BLAST page also provides options for changing the format of BLAST results.
Since we are not changing any format options, click on the  button on the formatting BLAST page, and a new browser window will open that contains the BLAST results. It may take a few minutes to generate the BLAST Results page.
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