Usually when you sequence bacterial genomes, the process of completely closing them (so-termed because most bacterial genomes are circular) whereby a single scaffold overlaps itself at the ends, requires a lot of effort. We’ve had very good luck with our Illumina sequencing and SPAdes assembly process, negating the need for things like primer walking, and we’re very close to finishing two very important genomes. Since this is unfamiliar territory for me (almost all genomes I’ve worked with thus far have been “draft” quality, usually with multiple scaffolds), I queried the Twitter micro community on the best methods for verifying a closed genome. This is from yesterday….
Enjoy some time pictures from time points 0200 and 0600 as we completed our 24 hour survey at the C transect site. The video I have uploaded is of the Platform and its fog horn that sounds every 20 seconds or so. Its a real joy to work and sleep to.
About a month ago, David Pitre (Thrash Lab undergraduate) and I went on our first excursion to collect Gulf of Mexico water samples as part of the LSU Culture Collection and surveying of the microbial communities of Southern Louisiana coastline grant. The Gulf of Mexico offers a vast realm of sampling sites for a microbiologist. Many sites around the Southern Louisiana coastline have never been characterized and are waiting for scientist like us to reveal their secrets and potential new uncharacterized microbes. One might say the Thrash lab motto may be “Gotta catch ‘em all”.
Like most great scientific expeditions, it started off with a change in sites. Though Louisiana does not suffer from a lack of water and coastline, roads leading to that coastline tend to disappear and end abruptly. So, with no road and a swath of bugs finding their way towards us, we decided it was appropriate to change our site from Freshwater Bayou (Kaplan, La) to the Calcasieu Lake Jetties (Cameron, La). An hour and a half drive east, and we arrived at the Calcasieu Jetties to an awesome view of the Gulf of Mexico and even some sandy beach!
Though the trip was for work, as a Northern kid, I always get excited seeing the beach and ocean.
But we had some water to collect, so we got the filtering things ready, got my waders on, and I headed out about a quarter of a mile into the Gulf to collect our first sample.
Once the samples were collected, they were brought back to our informal lab (the trunk of my car) and processed. Samples were collected for high thoughput culturing, cell counts, nutrients, and community sequencing (planktonic and particle associated). Samples were placed into our coolers and brought back to the lab for final processing.
Overall, it was a great trip and a great sampling site. Louisiana offers a beautiful coastline with ample sampling opportunities! Now I am back to continue to monitor isolates that we captured from the Calcasieu Lake Jetties sample. Here is to some exciting new cultures…Cheers!
Let’s face it. There are a lot of different ways to skin a microbe, and get the DNA and RNA out for whatever purposes you might deem necessary or useful. If you ask one or two people you’ll get one or two answers. What if you ask more than a thousand? Here is some insightful crowd-sourced feedback from Twitter on favorite and least-favorite methods of nucleic acid extraction, and we’ve done our best to include references with real comparative data, protocols, and applications:
Sometimes you want to collect samples in places that are hard to get to, or get out of. Sometimes those places are hot. Sometimes the people collecting your samples are not you, and they don’t have a lot of training. Sometimes there isn’t a freezer nearby. Or even electricity. Does this mean you can’t get nucleic acid samples from these places? Of course not. Here’s what Twitter had to say about this issue:
Technology is improving by leaps and bounds. With the current trends of massive, ultra-high-sequencing-coverage-combined-with-state-of-the-art-computational-binning-algorithms supported metagenomic assemblies and lasertweezer/microfluidics-enhanced, flow-cytometry based, MDA-powered single-cell genomics churning out incredible insights into the world of uncultivated microbes, it’s important to return, every once in a while, to the humble pursuit of good old-fashioned genome sequencing of simple, pure culture microbes. Here is a storify on just this from a recent Twitter exchange: