Comparing the new 16S rRNA V4 and ITS primers to the old primers-RESULTS!

The Knight lab has been working hard testing new primers for 16S rRNA amplicon production and its time to share our progress.

So far, the 16S rRNA V4 region forward primer (designated 515f) has been paired with five different reverse primers (806r, 926r, 967r, 1048r, and 1391r) to amplify ribosomal RNA from bacteria, Archaea, and Fungi.  Thanks to Jed Fuhrman and Amy Apprill, the 515f and 806r primers have also been modified, helping minimize the amplification bias against Crenarchaeota/Thaumarchaeota (515f) and SAR 11 (806r).  All primer pairs have successfully yielded PCR amplicons, and the amplicons from the 515f/806r and 515f/926r constructs sequenced.  The remaining primer pair constructs will be sequenced soon with an update to follow once we have the results.

The differences between the old and new 515f nd 806r constructs are described below:

Original 515f construct / modified construct (Jed Fuhrman, C to Y base change on the 5’ end)


Original 806r construct / modified construct (806rB, Amy Apprill, H to N base change mid-primer):


Why the new constructs, you ask?  And what does the added degeneracy mean?

  1. The barcodes, which were previously located on the reverse primer, are now located on the forward (515) primer.  This enables the user to pair the forward primer with various reverse primer constructs to enable longer amplicons.  We’ve tested the barcoded 515f primer with 806r and 926r.  Importantly, the barcoded constructs were screened in silico for secondary structure against a number of longer constructs (967r, 1048r, 1391r).  We have tested the performance of these constructs in PCR but have not validated the results on the MiSeq or HiSeq platforms.
  2. The degeneracy was added to the forward and reverse primers to minimize the bias against Crenarchaeota/Thaumarchaeota (515f modification) and the marine and freshwater alphaproteobacterial clade SAR11 (806r modification)

To compare the new primer constructs to the old ones and thus confirm the performance of the new constructs, we sequenced amplicons produced from both constructs applied to a number of studies.  Our intent was to sample a wide range of sample types to confirm that the new primer constructs produce data comparable to that obtained using the old constructs on a variety of sample types.  The studies/sample types that the constructs were tested on are:

-5 American Gut fecal samples

-5 American Gut skin Samples

-5 Body farm control Soil samples

-6 Body farm paired skin/soil samples

-33 Sloan house samples (various sites)

-15 Mouse decomposition control soils

-9 Rice Rhizome samples

-12 Agricultural soil samples

Below is a procrustes plot (using the unweighted UniFrac distance matrix on the left and the weighted UniFrac distance matrix to take into account taxa abundances on the right) comparing samples amplified using the original primer construct and the new, modified primer construct.  The calculated M2 value for the unweighted UniFrac based plot is 0.111 and for the weighted UniFrac based plot is 0.196.  With the exception of a few mouse decomposition and built environment samples, each sample produces extremely comparable results between the old and new primer constructs.  Importantly, very commonly studied sample types (stool, soil, skin) perform very well under the new constructs.




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What exactly is that sequencing data?

The idea for GenomePeek began two years ago when I was working with Karl Klose, Liz Dinsdale, and Rob Edwards to assemble a P. salmonis genome that was being particularly difficult, even though we had 9 gigabases of sequencing.   To check whether it was a single isolated genome I pulled out all the 16S reads that hit to 16S and then assembled them. All of the assembled contigs hit to P. salmonis, it was only until later that I found that genome had been shredded by an overactive transposon. We still haven’t solved that problem ….

Looks like someone did not do a complete bacterial isolation.

GenomePeek lay dormant for a year, until a student from SDSU’s bacterial sequencing class came looking for help. In the class the students were isolating a bacterium, sequencing, assembling, and then analyzing it.   The student wanted to create a recA phylogeny and was having trouble with taxonomic assignment.   Their question was: “which RecA gene do I use?”.   This set off a red flag immediately, since RecA is essentially a single copy housekeeping gene.   On inspection, their assembled genome did indeed have two full copies of RecA; one that hit to a Vibrio and one that hit to a Photobacterium.   At the time the two 16S rRNA genes hit to Vibrio, although they were clearly different from each other and hit to different species (since then a representative Photobacterium 16S sequence has been added to the NCBI 16S database that is now the top hit). It turned out that this bacterium also had two sets of other single copy housekeeping genes (I checked rpoB, groEL, nifD, gyrB, and fusA). One of each gene hit to a Vibrio species while the other was most similar to a Photobacterium species. Suffice to say the student was very disappointed after spending a few weeks analyzing and writing up a paper.   The idea occurred for a tool where one could submit sequencing data and then quickly get back a set of useful housekeeping genes for phylogenetic analysis. I thought this tool would save everyone’s time wasted on assembly, annotation, and analysis. By quickly checking sequences, we could easily detect whether the original sequencing data was contaminated. I wrote (more…)

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2014 Microbiology of the Built Environment Fellows

The Sloan Foundation has recently announced their 2014 Microbiology of the Built Environment Postdoctoral Fellows.   The awards, along with the titles of the projects are below.  Congrats all!   Look forward to detailed blog posts from all the awardees describing their upcoming projects.


Huan Gu at Syracuse, along with Dacheng Ran. “Understanding and controlling biofilms in the built environment

Sarah-Jane Haig at University of Michigan, along with Lutgarde Raskin and John LiPuma “Regulation of the microbial community structures in drinking water, from source to tap

Brian Klein at Forsyth Institute, along with Katherine Lemon “Microbiomes of indoor track facilities and runners who train indoors vs outdoors

Zachery Lewis at UC Davis along with David Mills and Katie Hinde (Harvard) “Role of the built environment as a venue for microbial cross inoculation between infants

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New Paper : On the intrinsic sterility of 3D printing

As a biologist with a 3D printer, one of the questions I get most often about 3D printed parts is, “Can you autoclave these things?” As it turns out, no, not really. There are only a handful of thermoplastics that can survive the autoclave process, and most of them are not very good for 3D printing. With few exceptions, only polypropylene and blends of polypropylene hold up to repeated autoclave cycles, and polypropylene is, unfortunately, very a difficult material to print. It shrinks a lot when it cools, which causes a lot of warping during printing, and it is very difficult to get molten polypropylene to bond strongly to cooler, solid polypropylene.

It turns out that this is all unnecessary. Fused deposition modeling (FDM) 3D printing involves shoving a rod of thermoplastic into a hot nozzle until it melts and squirts out the nozzle. For most popular 3D printing plastics like ABS and PLA, the nozzle temperature is somewhere between 180C and 260C, and the plastic stays at that temperature for around a minute, depending on what the toolpath looks like. It’s actually a lot like Pasteurization, except way overkill. Get it? Overkill?

Anyway, here’s how FDM 3D printing compares to various Pasteurization (in black) and autoclave (in red) protocols :

FDM 3D printing compared to pasteurization (black) and autoclave (red) protocols.


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“The Dirt on Antimicrobials”

Bill Walsh of The Healthy Building Network has posted a story on the subject,”the Dirt on Antimicrobials” that covers the health effects concerns from the chemicals themselves but does not address the currently popular subject of the health harm or benefits from the presence of and exposures to the multitude of microbes in, on, and around us.

The post starts out:

“The infusion of antimicrobial materials into building products is on the rise. Manufacturers now routinely add substances such as nano-silver and the pesticide triclosan to paints, tiles and grouts, carpets, solid surfaces, faucets, elevator buttons and toilet seats. The dirty truth is: they do not make people healthier. They do cause environmental harm throughout their lifecycle. And their overuse, like the overuse of antibiotics, may contribute to the evolution of microbes that are more resistant to our known antimicrobial defenses.”

See the complete story here.

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