Storify on finishing bacterial genomes

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….

Storify on microbial genome sequencing in 2014

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:

-jct

Recent Collaborative Thrash Lab SAR11 research

There are some exciting developments in the world of SAR11 microbiology, and I’d like to take a moment to summarize recent research I’ve been involved in. All three papers have come out this year in The ISME Journal.

circos3
The single-cell genomes from Thrash et al., 2014, aligned linearly according to scaffold size, showing (outside-in) GC content, scaffold boundaries, coding regions according to pan-genome status, and shared elements unique to the subclade Ic with orange lines

The final main project of my postdoc with Steve Giovannoni was a characterization of a deep-water subclade of the larger SAR11 group using single-cell genomics and metagenomics. This was a collaboration between our lab and that of Ramunas Stepanauskas and Ed DeLong, and although it began at Oregon State, the work carried over (like many projects do) into my appointment at LSU. The main findings of the paper were that this “subclade Ic” dominated deep-water metagenomic reads compared to extant surface-originated genomes, and that a few gene-content specific variations could be identified that may distinguish these organisms from the surface strains, but that, on the whole, the main observable differences were in global genomic footprints such as expected average genome size, average coding region size, intergenic spacer size, and preferential amino acid substitutions. The deep water ecotype, subclade Ic, has obviously been evolving separately from the surface subclades for some time based on these accumulated variations and phylogenomic branch lengths, however, it also appears these organisms share a similar metabolic niche with the surface strains: they are predicted to be obligate aerobic organisms with a particular appetite for common metabolic intermediates found in the cellular milieu of most organisms: simple carboxylic and amino acids and C1 and methylated compounds.

Read more here: Thrash, J. Cameron, Ben Temperton, Brandon K. Swan, Zachary C. Landry, Tanja Woyke, Edward F. DeLong, Ramunas Stepanauskas and Stephen J. Giovannoni. (2014) Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype. The ISME Journal. doi:10.1038/ismej.2013.243.

 

Steve was also invited to write a Winogradski Review article in coordination with him recently being given the prestigious Jim Tiedge Award from ISME. Ben Temperton, who is also a former postdoc of Steve’s, and who worked closely with me on the subclade Ic study, above, was a co-author. Steve took the opportunity to review many of the observed variations between streamlining selection on free-living, small genomes like SAR11 and Prochlorococcus, and the small genomes of obligate intracellular symbionts like Rickettsia. Importantly, the review also pointed out that examples of streamlining selection are becoming more prevalent as data from single-cell genomics and assembled metagenomes continues to pour in. Indeed, the odd nutrient requirements that are sometimes a result of streamlining selection may be at the heart of the difficulty in cultivating many of these organisms in the lab.

Read more here: Giovannoni, Stephen J., J. Cameron Thrash, and Ben Temperton. (2014) Implications of streamlining theory for microbial ecology. The ISME Journal. doi:10.1038/ismej.2014.60

 

Dovetailing nicely with the theoretical concepts brought up in Steve’s review was a third piece from our lab, a detailed study on an unusual vitamin requirement in SAR11 that was discovered by Paul Carini, currently postdoc-ing with Alyson Santoro. This work identified that members of many SAR11 subclades are unable to make the vitamin thiamine by typical synthetic means, because they lack a single gene in an otherwise complete pathway, thiC. As a result, they are unable to synthesize 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), a direct precursor to thiamine. However, Paul also discovered that SAR11 has the capacity to use free HMP in media to relieve thiamine limitation, and identified a putative transporter for HMP in several SAR11 genomes. Additionally, HMP measurements from the Sargasso Sea and cultivation experiments pointed to a possible natural source of HMP from cyanobacteria, thus providing an explanation for how SAR11 can remain successful without the key thiC gene.

Read more here: Carini, Paul, Emily O. Campbell, Jeff Morré, Sergio Sañudo-Wilhelmy, J. Cameron Thrash, Samuel Bennett, Ben Temperton, Tadhg Begley and Stephen J. Giovannoni. (2014) Discovery of a SAR11 growth requirement for thiamin’s pyrimidine precursor and its distribution in the Sargasso Sea. The ISME Journal. doi:10.1038/ismej.2014.61

-jct

Last #LSUCompBio seminar of the semester

I’m giving the final #LSUCompBio talk for this semester (so I won’t be live tweeting it, most likely) tonight at 5:30 in LSA A101. The title of my talk is “We’re not in a petri dish anymore: pulling back the curtain on microbial genomics using cultivation-independent techniques.” The talk will be broadcast live and archived afterwards. I’ll be giving an introduction to microbial metagenomics and single-cell genomics, with an example of combining the two from a recent project of mine.

-jct

Computational Biology Seminar Series starting this semester

Hey LSU students, there is going to be a Computational Biology Seminar Series this semester where you can hear researchers from LSU who are involved in many facets of computational biology discuss their work and specific techniques they use. More info at the seminar series website and also at CCT. Check back there frequently (linked above right) as more information will become available, including the schedule for additional speakers (like me!).