Finally, an update - huge batch of sequence data, part 2

Hi everyone,

So, it's been about two and a half months since I last opened up this Experiment page and published any new information on the progress of the project, so I figured that March is as good a time as any to get back into it.

Major updates?

With regards to algal cultures...they have sadly not fared too well. A wave of mold took over many cultures, starting with JIAC33 as mentioned in my last lab note. In addition, a good number of other ones have simply fallen to other forms of contamination. This, I knew, would be inevitable - although I did get antibiotic therapy to successfully clean one batch of algae with which I prepared many invaluable DNA templates, the cultures just regained their bacterial contamination over time; such is the difficulty of home research. (JIAC7, in particular, always harbored "buddy bacteria" that were impossible to remove, either with the use of antibiotic media or streak isolation.) In addition, I noticed that some other cultures, after routine observation of colonies, would mysteriously switch species. Funnily enough, it was almost always from something else to Scenedesmus, the latter being really weedy, hardy and quick-growing. The same attributes that make species of this genus ideal candidates for biofuel or bioproduct research are area nightmare to deal with when trying to maintain higher-throughput cultures of many individual and diverse species. So I lost most of my Selenastraceae cultures in this way, as well as JIAC5 and JIAC7 (once there were no more bacteria in this culture, I knew something was up). And then, some strains just....died out. Perhaps they were more fastidious and required micronutrients that I simply never provided in my cultures at a high enough concentration to sustain them, so the populations of cells just got more and more sickly until they decided "hey, we're not going to reproduce anymore" and then, they became non-culturable and non-transferrable. My trebouxiophytes, JIAC13, 14, and 13i, especially suffered from this problem (which makes sense, they being adapted to living in other cells - probably got some juicy amino acids or other supplements that they stopped synthesizing on their own and that I didn't expect them to need), but so did JIAC30, a Selenastrum (as far as I know now). I mean, it's been hard - maintaining lots of species of algae indefinitely is a difficult task for professional culture collections with decades of experience, much less a high school student in a home setting. (I can't hire an undergrad to help me with my research....I am the undergrad.) But that's no excuse, and I won't let it stop me from continuing my scientific endeavors through senior year.

So on the bright side....all of my Scenedesmus have thrived, which is something, at least. The Chlamydomonas I cultured from school are all good too, although I suspect they are not C. reinhardtii but rather C. moewusii or some other species, due to their seeming inability to grow heterotrophically on acetate. What else....I saved one copy of the suspected endosymbiont Chlorella and most of the Volvocales (JIAC4, 12, 15, and 26, although 12 and 26 seem to be at low densities and might die out soon). This handful I hope to give to a culture collection eventually or keep for my own research. However, for my project at hand, I gotta work basically with only the digital data I have and that's it. Which is kinda...not good, as being able to examine microscopically living cells of different cultures would have been a huge component of the project. Without this, there's no way of "double-checking" the accuracy of DNA identifications, especially given the current limitations of databases and the literature (the improvement of which was supposed to be a goal of my project, and not a caveat of its legitimacy). So that's decidedly not good, but at the end of the day, I'll work around it. I've still managed to get hundreds of new algal DNA sequences, which I will upload to the public domain (GenBank), and I can cross-check tentative identifications with multiple barcode regions to improve my confidence in them. Perhaps a final detailed study will be narrowed in scale to the Scenedesmaceae cultures I still maintain and can perform microscopy with, but I won't discount the data I've already managed to collect and its potential to help the current state of the field. Every little bit helps.

Which, despite my huge digressions, is the whole reason I'm writing this today. After a lot of delays in running PCRs, preparing plates, pipetting primers and packaging shipments to the University of Arizona, I finally managed to get all of the data downloaded from round 2 of sequencing today! Although I don't know that the UA Genetics Core follows my project here, I owe it a huge thanks regardless for the invaluable role it has played throughout the progress of this project. Not only have its team members been extremely informative and accommodating to my youngling research needs and inexperience, but they are always kind and supportive as well. The kind of moral support and flexibility they've given me is not part of any job description, so it means everything to me that they seem as committed to my success as I am. Even though I am a first-time researcher in this field, still teaching myself the fundamentals of genetics and making all the rookie mistakes, they have given me the respect of any well-tenured, world-class scientist, and have always helped give me confidence in my ability to contribute to the field. So once again...thank you.

 

My Sample Lifecycle Manager homepage - now with two new completed plate sequencing orders.

SO! Data round 2 - I ran the sequenced PCRs almost entirely from antibiotic-treated, peak-healthiness algal cultures that I first prepared and harvested last fall, so I have very high hopes that the bacterial contamination which corrupted my previous attempts at tufA and UPA data will not be an issue here! In addition, I reran some troublesome or missing rbcL, 18S, and 26S reactions, so hopefully they will turn out neatly. ITS....has always been a problem, but with this new round, I've gotten at least a couple more representative sequences from different algal species, so we'll see how that goes. At this point, I only have the raw files downloaded. In the coming months, my work for this project will be almost entirely digital - renaming and examining files, editing sequences, piecing them together, and only after all that, finally performing the meaty phylogenetic analyses. So the next time I post a lab note will be to provide a preliminary update on the number of sequences which have turned out to be readable, and with which I can then proceed to the editing step. After that, I'll aim to provide regular updates on the sequence editing/base-calling progress (the worst step, by far) and any phylogenetic experiments I might run as well.

Thanks as always for all of your support as well. Yes, you! Sorry if this project has been dormant for too long, but....Idunno, that's how life works, I guess. If science is my life, I guess it's subject to the same ups and downs, moments of high activity and times of rest. But now, at least, stuff's happening. So stay tuned - and let's keep exploring.