Terence Crofts
banner
terencecrofts.bsky.social
Terence Crofts
@terencecrofts.bsky.social
🙌Congrats to Hayden and Elizabeth for their co-first author publication and thanks to our collaborators Erica Hartmann at Northwestern and Frank Oliaro at the Shedd aquarium (and to Zymo research @zymoresearch.bsky.social for the sample human fecal material!)
September 11, 2025 at 3:56 PM
Why does this matter🤷‍♂️?

We think it is important to find resistance genes from other environments before they reach the clinic🏥or farm🐮. Functional metagenomics allows us to do this, and our method, by using less DNA more efficiently, opens up many new microbiome samples!
September 11, 2025 at 3:56 PM
Notably, simply sequencing metagenomic DNA would not have let us discover these functions, highlighting the importance of the *functional* aspect of functional metagenomic libraries in finding new resistance genes
a pikachu wearing a detective hat is holding a magnifying glass .
Alt: a pikachu wearing a detective hat is holding a magnifying glass .
media.tenor.com
September 11, 2025 at 3:56 PM
With just ~30 and ~100 ng of DNA, both microbiomes gave good libraries!

We used antibiotic selections💊 to link unannotated❓MFS transporters from the aquarium to tetracycline resistance and a new acetyltransferase (we call it SatB) from the swab to streptothricin resistance.
September 11, 2025 at 3:56 PM
We used our approach to tackle a pair of low biomass microbiomes that would usually be difficult to study via functional metagenomics: an aquarium microbiome🐟and a fecal swab💩.
September 11, 2025 at 3:56 PM
We show that we can start with 10- to 100-fold ⬇️input metagenomic DNA (10s to 100s of nanograms) and still make ~gigabase libraries without needing to rely on potentially biasing DNA amplification.
September 11, 2025 at 3:56 PM
We previously found that METa assembly libraries can be made with ⬆️efficiency than classic methods resulting in libraries containing 100s of gigabases of DNA.

Here, we answered 'what about making libraries from limited samples?' 🤏

journals.asm.org/doi/10.1128/...
Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes | mSystems
Bacterial genes in microbial communities, including those that give resistance to antibiotics, are often so novel that sequencing-based approaches cannot predict their functions. Functional metagenomic libraries offer a high-throughput, sequence-naive solution to this problem, but their use is often held back due to their need for large quantities of metagenomic DNA. We demonstrate that our functional metagenomic library preparation method, METa assembly, can prepare these libraries using as little as ~30 ng of DNA, approximately 1,000-fold less than other methods. We use METa assembly to prepare functional metagenomic libraries from low-biomass aquatic and fecal swab microbiomes and show that they are home to novel tetracycline efflux pumps and a new family of streptothricin resistance gene, respectively. The efficiency of the METa assembly library preparation method makes many otherwise off-limits, low-biomass microbiome samples compatible with functional metagenomics.
journals.asm.org
September 11, 2025 at 3:56 PM
We make🛠️these libraries via METa assembly (previous twitter thread👇) using tagmentation to fragment DNA/add mosaic end tags on all fragments. This lets us use assembly cloning to *efficiently* ligate fragments into plasmids with a mosaic ends cloning site.

x.com/TerenceCroft...
Terence Crofts on X: "I am thrilled that my METa assembly paper with collaborators @ericamhartmann and @alexmcfarland_ is online now at @mSystemsJ: https://t.co/Na6iDJTnYV We show that functional metagenomic libraries (FMLs) can be made using 20-fold less DNA and with 80-fold greater efficiency 1/n https://t.co/3MO7IgScNY" / X
I am thrilled that my METa assembly paper with collaborators @ericamhartmann and @alexmcfarland_ is online now at @mSystemsJ: https://t.co/Na6iDJTnYV We show that functional metagenomic libraries (FMLs) can be made using 20-fold less DNA and with 80-fold greater efficiency 1/n https://t.co/3MO7IgScNY
x.com
September 11, 2025 at 3:56 PM
Swenson 2018 tested a few pressure cookers for this and found instant pot can kill spores pretty well. I’ve been using one in lab without any issues for a couple of years

pmc.ncbi.nlm.nih.gov/articles/PMC...
Assessment and verification of commercially available pressure cookers for laboratory sterilization
Laboratory science requires careful maintenance of sterile reagents and tools as well as the sterilization of waste prior to disposal. However, steam autoclaves typically used for this purpose may not...
pmc.ncbi.nlm.nih.gov
June 23, 2025 at 12:31 PM