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Columbia U Researchers Develop NGS-Based Spatial Metagenomics Method

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NEW YORK – Researchers have developed a new droplet-based sequencing-based method for extracting information about spatial relationships between bacteria in microbial communities. 

Led by first author Ravi Sheth and senior author Harris Want, of Columbia University Medical Center, the researchers combined next-generation sequencing with plot sampling methods used in ecology to analyze microbiomes at micrometer-scale resolution. 

The method, called metagenomic plot sampling by sequencing (MaPS-seq), showed that some bacterial species are more likely to colocalize with each other in the mouse gut microbiome. It also helped show that those associations change in response to dietary perturbation. The researchers published their results today in Nature Biotechnology.

"This provides a higher resolution of microbiome organization and creates useful biomarkers for changes in the microbiome you're not going to be able to get looking at a bulk composition," Wang said. "We think it's going to be important for delving into mechanistic studies of the microbiome."

The researchers said their approach is similar in principle to inDrops, an indexed, droplet-based, single-cell sequencing method developed by Harvard University researcher Allon Klein. Both sequencing methods use custom microfluidics and encapsulation technologies. 

The researchers have applied for a patent on MaPS-seq and said it could be used to identify biomarkers related to changes in microbiome composition, or commercialized as a kit. The method uses custom barcoding beads and microfluidic chips, whose designs have been made public.

"I think it's a great step forward," said Justin Sonnenburg, a gut microbiome researcher at Stanford University who was not involved in the study. Gut microbiomes are complex ecosystems that researchers are increasingly trying to understand spatially. MaPS-seq "allows investigators now to take the next step and ask questions about what is functionally happening within these neighborhoods," he said.

The method uses a new library preparation process that fixes microbes in a gel to preserve the spatial relationships, then shatters that gel into tiny pieces so the bacteria within them can be lysed, barcoded, and sequenced. Encapsulation beads contain barcodes tagging all bacterial genomes within a particular gel shard, as well as 16s ribosomal DNA primers to identify the species.

Sheth said they haven't calculated exact costs yet, but said it was "on the order of hundreds of dollars per sample," mostly due to the cost of Illumina sequencing.

MaPS-seq would likely be a cheaper, higher-throughput method of resolving complex communities than current imaging techniques like laser-capture microdissection or fluorescent in situ hybridization, said Carolina Tropini, a microbiome researcher at the University of British Columbia (UBC) who was not involved with the study. But she suggested that researchers use both approaches.

MaPS-seq, she said "needs to be supplemented. While you do get spatial characterization, you lose the larger scale, you don't have any information about where the droplets come from."

The researchers acknowledged that their method does not reconstruct a 2D or 3D physical map of the community. "You destroy all the global features, but you retain the local features that tell you about associations," Wang said. "If you sampled enough of these clusters you might be able to reconstruct some version of the [physical map], but that's not something we demonstrated. The idea is that we're also sampling a small number of cells compared to the total number present."

Wang compared the approach to the way ecologists establish relationships between species in larger-scale ecosystems, like forests. "If a tree is always next to another shrub that's an ecologically relevant community. That's what we're trying to get at," he said.

The researchers performed proof-of-concept studies on gut microbiomes, but "one could imagine using this with many types of communities," Sheth said.

Wang noted that their method provides information that isn't captured by the diversity of organisms or their relative abundance — until now the two main ways researchers have looked at microbiota.

The new data shows that the distribution of bacterial species in the mouse gut is not homogenous, the Columbia researchers said, and that certain species are more likely to be neighbors than others. They also performed studies before and after a change in diet and showed that the perturbation led to spatial restructuring, along with expected changes in diversity.

All the researchers suggested that adding the ability to sequence transcripts, rather than just the 16s identifying regions, would take the method to another level.

"If you're able to tell at one part that things are metabolically different, that would be really important," UBC's Tropini said.

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