Mic 433, Microbial Genomics
Lab exercise #2
Due date: Tuesday January 27 Name:
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. 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 clicking on the “Alignment
Parameters” before you assemble. What does setting the “Minimum Match
Percentage” to 100% do when you try to assemble the sequences? Why?
2. Assemble your sequences using the default Alignment Parameters. What is the average coverage on the consensus?
3. 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?

4. Identify the problematic base calls on the consensus sequence and try to correctly edit them.
5. Trim the ends of your consensus sequence both by hand using the chromatographs as a guide, as well as the “Trim Ends” function. How many base pairs long is your gene after it is trimmed?
6. Why does the quality of the sequence for the reactions primed with 1104R or 005F start to decrease after several hundred base pairs?
7. Print out a copy of your consensus sequence in Genbank format and highlight the sequence of primer 357R (5'-CTGCTGCCTCCCGTAGGAGT) .
8. What gene does your consensus sequence code for? From what organism was it obtained?
9. 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?

10. 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?
11. If you wanted to clone the sequenced fragment into the cloning vector "pTrueBlue" what restriction site could you use to retain as much of the fragment as possible?
Highlight these restriction sites on the printout of the consensus sequence.