TARGETING THE DUCKWEED MICROBIOME FOR PLANT HEALTH AND SUSTAINABILITY OF NATURAL RESOURCES

<br>What was accomplished under these goals? In 2015 the first Oxford Nanopore Technologies (ONT) palm-sized MinION nanopore DNA sequencer became commercially available. Over the last several years significant reductions in the sequencing error rates, and increased flexibility in the DNA library preparation reagents, have made MinION nanopore sequencing a promising option for many research labs. We tested the MinION nanopore sequencer to analyze duckweed microbiome DNA samples. The initial pilot runs in our lab were successful and we are in the process of switching to carrying out much of our duckweed microbiome sequencing using the MinION sequencer. Sequencing microbiome DNA at commercial labs using Illumina instruments costs $50-$75 per sample and takes 3-5 weeks to receive the sequence data. By switching to the MinION system, we can reduce the cost to $10 per sample and have the microbiome sequence data within 48 hours. The MinION system produces long DNA reads, and in our application, we can generate 1,600 bp 16S gene reads, as opposed to the 250 bp 16S reads using Illumina. The greater length in reads will allow us to better characterize the bacterial complexity in our samples. The MinION system will allow us to analyze 5x more microbiome samples for the same cost, and will speed our research progress. Because all the work is done in our lab, using the MinION nanopore sequencer gives us the ability to customize all steps in the process, from DNA library preparation to data output, in a way that is best suited to our work with duckweed microbiomes. In our initial MinION runs we used the ONT 16S-barcoding kit to generate the duckweed microbiome DNA libraries for sequencing. In this protocol 16S universal PCR primers containing nanopore adapter sequences are used to amplify the 16S gene from the duckweed microbiome template DNA. While the libraries we generated worked and we generated ~100K high quality 16S amplicon DNA reads (~10K reads per barcoded sample), some barcoding reactions failed. Using the 16S-barcoding kit, the barcodes are added during the PCR amplification step, and if the PCR amplification fails and produces little or no amplicon DNA, the barcodes are wasted. Since the barcoded primers are the limiting reagent in the ONT 16S-barcoding kits, this translates into fewer samples that can be analyzed. To avoid this problem, developed a protocol using the ONT native, ligation-mediated, barcoding kit. The native barcoding kit uses an enzymatic ligation step, as opposed to PCR, to add the barcodes and adapter sequences to existing PCR amplicon DNA or genomic DNA. Using this approach, the sequencing barcodes can be added to each 16S amplicon sample after the PCR step, giving us a chance to check the quality of the PCR step before adding the barcodes. In cases where the PCR step does not produce sufficient amplicon DNA, we can repeat the amplification step until it works, or drop that sample from the experiment. This approach eliminates cases where barcodes are wasted on samples lacking high quality amplicon DNA. The native barcode ligation strategy can also be used with any DNA amplicons, not just 16S amplicons, as well as being used on unamplified metagenomic DNA for whole metagenome shotgun sequencing applications. We have isolated as pure cultures, and identified by 16S gene sequencing (long Sanger reads from GeneWiz), 80 bacterial strains from duckweed microbiomes that were collected in and around New York City (primarily from duckweeds found in Prospect Park, Central Park, and Alley Pond Park). Using bacterial genome databases, we have noted a few promising bacterial strains in our collection that are likely to have antibiotic degrading potential. This is based on the presence of predicted degradation pathways within their genomes, or based on their close relatedness to similar bacterial species that have been reported to degrade antibiotics. <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? We have made advances in duckweed microbiome analyses using QIIME2 and additional analysis tools available through Microbiome DB. These advances will allow us to characterize and compare duckweed microbiomes with greater efficiency and greater flexibility. Our current data suggest the the duckweed microbiomes that form naturally in duckweeds isolated from NYC, and those that form on sterile duckweeds, colonized from microbiome donor sources (laks and pond water and sediments) are similar to other published duckweed micrbiomes in their bacterial compostion (at the level of class). We have preliminary results showing the antibiotic stressors alter the composition of microbiomes that are able to assemble on duckweeds. The diversity, as measured by Faith's PD, is lower on duckweeds where the microbiomes assembled in the presence of antibiotics in the water. We are in the process of repreating these experiments. <br><br><b>Publications</b><br>

<br>What was accomplished under these goals? Work on manipulating the duckweed microbiome has begun (in the PI's lab at Brooklyn College). The PI has made some progress in colonizing sterile laboratory duckweed from natural microbiome sources. Progress has also been made toward creating a "synthetic" duckweed microbiome from cultured bacterial strains isolated from natural duckweed microbiomes. The COVID-19 pandemic has slowed laboratory research. The PI was able to begin lab research in the spring of 2021, and students have been able to begin working in the lab as of September 2021. However, the density of students that can work in the lab is at the same time is about ~70 capacity. <br><br><b>Publications</b><br>