Revolutionizing Microbial Sequencing: UCSD Scientists Develop New 16S rRNA Method Using Acoustic Liquid Handling

Written by Bioscribe on December 20, 2018

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When your sample sizes range in the thousands, the cost of library preparation can be critical. Miniaturization and automation have proven to be game-changing for scientists using costly whole-genome sequencing and cDNA synthesis methods. But what about other widely used molecular biology methods, such as 16S rRNA and 18S rRNA amplicon sequencing?

A team of microbial ecologists from the University of California San Diego and the Scripps Institution of Oceanography in La Jolla recently developed a miniaturized high-throughput amplicon library preparation method with the Echo® 550 Acoustic Liquid Handler and reported significant cost savings.

As described in the new publication in the American Society for Microbiology journal, Rob Knight, Jeremiah J. Minich and colleagues were able to reduce library preparation costs to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, “without compromising sequencing success or distorting the microbial community composition analysis.”

With such reductions in reagent costs, the Earth Microbiome Project, the largest crowd-sourced microbiome project, would have saved $500,000 to $1,000,000 in library preparation costs alone, enough to cover the entire sequencing costs for the project, the authors noted.

Building on the globally recognized protocols for 16S rRNA sequencing adopted by hundreds of labs in the EMP, the UCSD team set out to develop a low-cost 384-sample miniaturized (5-µL volume) amplicon library preparation protocol and demonstrate its utility.

They evaluated the effects of reducing PCR volumes and varying input genomic DNA (gDNA) volume on microbiome signatures and validated the method by sequencing microbiome samples from five body sites of 46 individuals of the marine fish Scomber japonicus (Pacific chub mackerel) across seven months of sampling.

Fish microbiome samples were processed with the miniaturized 5 µL reaction volume and 0.2 µL of gDNA, as well as the standard 25 µL reaction volume with 1 µL of gDNA. Comparing the two, the team found that the miniaturized 5 µL-volume protocol performed as well as the standard 25 µL-volume protocol.

“Our results demonstrate that the miniaturized PCR protocol maintains data quality with respect to traditional methods while achieving substantial cost advantages and producing much less plastic tip waste,” the authors wrote.

The use of an acoustic liquid handler also permitted dramatic decreases in sample volume usage, which may be important when using precious historical samples with limited volumes or running large numbers of assays per sample.

To read and download the full paper, please visit ASM here.

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