433, Mic Microbial Genomics Lab exercise #3 Due February 2nd or 4th
Name: Mary Ellen Hoinski
The goal of this session is to gain some practical experience in assembling and analyzing a consensus sequence, and to solidify your understanding of DNA sequencing chemistry. The contig you build will be assembled from individual sequencing reactions that were performed by a commercial sequencing center.
1. Launch the Sequencher application (found in the applications folder). 2. Choose the Import command from the File menu and choose Sequences. 3. Change the Files of Type to All Files, and select the folder of sequences on the computer (the file is on the server and entitled 14 sequences under MMG433 Lab 3-). Select all of the sequence files in the folder, and click Open. 4. A project window will appear with the individual sequence icons highlighted. 5. Select the Assembly Parameters button to view the criteria being used to assemble your consensus sequence. Click OK to assemble your sequences using the default Alignment Parameters. Then click on the Assemble Automatically button (make sure that all of you sequence icons are still highlighted). A contig sequence will be generated. Any sequence data that could not be assembled in the contig sequence will still be present as a sequence icon in the project window. 6. Different criteria can be used to assemble your sequences- ranging from liberal (will align more dissimilar sequences) to very conservative (only aligns perfect matches). Try out the various options for aligning your sequences by dissolving your contig by using the Dissolve Contig command from the Contig menu. Then change the Assembly Parameters and click on Assemble Automatically (as in step 6 above) to construct a contig with the new parameters. What does setting the Minimum Match Percentage to 100% do when you try to assemble the sequences? Why? 1) I received 10 fragments and a contig of 2, there were only two sequences that matched 100 percent so the other 10 had some variation 7. Reconstruct the contig using the default parameters (85% Minimum Match Percentage, 20% Minimum Overlap). Open a contig editor by double clicking on the contig icon. What is the average coverage on the consensus (Note there is no feature in the program to calculate this for you. Determine the amount of coverage
from 4-5 points throughout the length of the contig and take the mean)? Why is average coverage an important piece of information? 1) My average coverage was 4.8 (The mean of 4,5,5,6,4) If at a given point I picked, there were six overlaps that means that at the base pair there are that many overlaps. A higher coverage is good because it means that there is more overlap between the sequences. 8. How many individual sequence reactions were performed to create your final contig? How many reverse and how many forward primers were used in the contig assembly? What is the difference between primers that are designated forward (F) or reverse (R) primers? 1) There were twelve sequence reactions performed, one reaction for each sequence 2) 6 reverse and 6 forward primers 3) They go in different directions and are reverse complements of each other 9. In the VIEW menu select Bases, Maps, Overview and then Bases. The lowest sequence is the consensus sequence generated from the alignment. Click on an N or one of the ambiguous bases in the consensus sequence. Then click the Show Chromatograms button. If you cannot see the peaks, adjust their heights by using the slide bar on the left hand side of the screen. 10. Correctly edit 5 ambiguous bases throughout the entire consensus sequence. (simply type the correct letter to replace those highlighted) . 11. Why do problematic base calls arise, and how you did resolve them? Were there any that could not be resolved? 1) Problematic base calls arise because there are too many peaks in the spot that the computer has trouble deciphering which the correct peak should be for the base pair. Towards the end of some sequences there was not enough data to resolve the problematic base calls 12. Once youve cleaned up some of the ambiguous base calls, click on the Contig menu and select Create New Seq from Consensus This will create a file of your sequence in Genbank format. 13. Now we will let Squencher clean up the sequences. Sequencher allows you to trim ends based on either the frequency of ambiguous base calls at the ends of the sequence, or it the Phred data is available, by the frequency of low confidence base calls. Dissolve the contig as in step #7. Then, from the Sequence menu, choose Trim Ends. An Ends Trimming window will appear. Check the automated trimming calls for each of the sequences by double clicking the sequence icon on the left.
Sequence that is colored red will be trimmed off. Click on Show Chromatograph to view the raw data. You can alter the trim by checking or unchecking the 5 and 3 boxes by each sequence. 14. Why does the quality of the sequence for the reactions primed with 1104R or 005F start to decrease after several hundred base pairs? 1) The dideoxy stops the fragments as the strand gets longer and that is why there is a decrease after several hundred base pairs. The quality greatly decreases and the computer cannot continue reading 15. After how many bases does signal intensity drop off in the sequencing reaction that was primed with 357R? Why is there such a dramatic drop off in the signal intensity? 1) Approximately 300 base pairs is when the signal intensity drop off, this is because it reaches the end of the template and there wasnt enough for the primer to read 16. If the sequence of a 16S rDNA gene primed with 357R continued beyond 400 bases without a decrease in signal intensity, what type of template do you expect was used for sequencing? 1) This is probably because its a circular template instead of linear 17. Additional criteria for trimming can be accessed by clicking on the Change Trim Criteria button. This will allow you to change the number of consecutive ambiguities or percent PHRED confidence that a sequence must meet in order to be incorporated into the consensus. 18. Select the Trim Checked Items button in the Ends Trimming window, and Trim your sequences. 19. Return to the original window and Assemble Automatically the contig with the trimmed sequences. How many base pairs long is your gene after it is trimmed, and what is the length difference between it and the untrimmed sequence you manually edited? 1) 1453bps, my original was 2056 so there is a difference of 603bps 20. Edit your sequence manually as before (step 10-11), but this time try to correct all of the ambiguous bases. Once finished, Create New Seq from Consensus as in step 13.
21. Open your newly created sequence file. Click on find and search for the sequence of primer 357R (5'-CTGCTGCCTCCCGTAGGAGT). Where is it located in the sequence? 1) Between 1143 and 1162 22. Click on Cut Map to see an overview of the restriction enzyme sites on your sequence. Note that you can add or remove restriction enzymes by clicking on Select Enzymes, and you can click on Options to select unique cutters (cut the sequence only once), 2-cutters, 3-cutters, and so forth. 23. If you wanted to clone the sequenced fragment into a cloning vector what restriction sites could you use to retain as much of the fragment as possible? What would be the size of the fragment? 1) Kpnl would give the largest fragment possible because it cuts at 1040* 2) 845