Lynna Ye - Week 6: My Last Week at the Ware Lab

I have completed my lab experience at the Ware Lab in Rutgers Newark!

This last week I worked on finishing up my project, which though ultimately unsuccessful, was still a thrilling achievement. Dr. Ware drove away to her hometown of Canada and most of the grad students were busy running the high school program, so it was very quiet in the lab, with only grad student Dominic working at his desk, much like my first week. The only difference was that he had started to leave his laptop and do some work at the benches where I was, which was an exciting change. On the other hand, I began to spend more time on the computer doing data analysis.

On Monday, I used NCBI's Blastn to compare the sequences I received from Friday''s PCR against others in the database. Unfortunately, the results all suggested similarities with genes from Homo Sapiens instead of species from Odonata (dragonflies or damselflies), which means that the DNA may have been contaminated, or that there was a low yield of PCR product. After blasting all my sequences, I was disappointed to discover that only one sequence yielded similarities to some species of dragonflies. While I was fairly upset that my sequences were not successful, I was very relieved that one worked. The rest of the day, I started doing my last batch of extractions and tried out the programs I would have used for my analysis.

Tuesday, I finished extracting the DNA from the last 32 samples from nearly 500 legs, which felt like a great achievement. As I was doing this, I ran a few more PCRs in hopes of getting a few more samples sequenced to add to my analysis. I tried the usual troubleshooting methods again - adjusting thermal cycler temperatures and PCR recipes, but alas I was met again with more failure. Dominic told me that I should probably stop trying to do PCRs, since I would only be here for a few more days and they were not likely to work. So, I decided to re-PCR all the samples that had previously worked to try and sequence again for a hopefully better result, and to start my analysis with the one eligible sequence that I acquired as a sort of simulation for what I would have done had I had more data. However, I encountered some troubles at this stage  as well. I discovered that the primers I used in my PCR, TL2 and C1J, amplified a different region of the COI gene than most of the other sequences I downloaded from GenBank, and as a result it was difficult to align all the sequences for comparison. To combat this issue, I downloaded some longer COI sequences that included both regions of the gene, so that sequences from different regions of the gene could be aligned with each other.

Brightly color-coded nucleotide bases assisted in alignment. Notice how some sequences at the bottom do not begin until most others have already ended.

Wednesday, I focused on making a phylogenetic tree, which would in theory tell me what species my mystery sequence was by grouping my sequence on a sister branch with another previously identified sequence from the database. After aligning the sequences with ClustalX, I had to do manual alignment in Mesquite to make sure that the complementary portions of each sequence were lined up with each other. Then, I ran GARLI, which uses a complicated algorithm and many generations of repetitions to create a "best tree". After this was complete, the same program was used to create bootstrap replicate trees, which determines the strength of the branches created in the "best tree" based on probability of two branches being found next to each other throughout the replicates. While the program was running, I worked on my poster. I would like to give a big thank you to Dominic for giving me PCR advice, showing me how to use all the complicated programs, and reading over my poster!

Thursday was the last day of my 6-7 week adventure at the Ware Lab. Dominic helped me to combine the bootstrap and the best trees to create my final tree.

This tree suggests that my sequence belongs to Cordulia aenea. The percentages on the branches reveals the support for each.

Since I was pretty much done with my project (although I was not successful in collecting much data), I helped out some grad students, first organizing Dominic's vast collection of beetles and other bugs, then joining undergrad Miriam with scanning the wings of dragonflies that Melissa and Will caught in Wisconsin so they can improve the program they are working on to identify species through wing venation alone. I made sure to personally thank and say goodbye to all the grad students, and after I organized my many boxes of DNA extractions and legs for someone to possibly use in the future, I left the lab for the last time to catch my final train ride home.

Overall, I had a very robust and enjoyable lab experience. I'm glad to have met so many wonderful people - the friendly undergrads, the brilliant grad students, and the fabulously kind PI, Dr. Ware - who I all hope to see again in the future. I've learned so many things about lab life (and public transportation) and I'm very happy to have spent my summer at the Ware Lab!

Thanks for reading,

       Lynna Ye