Category: Automation and High-Throughput Technologies
Our understanding of the microbial communities in human health, metagenomics, and metataxonomics has been growing rapidly in recent years and is being increasingly elucidated every day. Amplicon sequencing of highly conserved prokaryotic 16S ribosomal RNA (rRNA) regions has long been the standard technique used to assess the diversity and phylogenetic classification of these communities. While advances in next-generation sequencing are enabling routine whole-genome shotgun sequencing in microbial communities, 16S rRNA amplicon sequencing is still frequently used for quick diagnosis of samples. Here, we perform a standard Illumina 16S rRNA amplicon sequencing library preparation at miniaturized scale using the Labcyte Echo® 525 Liquid Handler, effectively reducing reaction volumes and input sample while maintaining sufficient read depth to accurately capture the community. The result is a significantly cost-reduced workflow per sample, saving reagent costs, operational time costs, and valuable sample DNA. We found that this miniaturized process produced plenty of amplicon library for downstream sequencing. Furthermore, decreasing the sample input DNA did not influence our results and analyses, all conclusions stayed consistent despite reducing the input by 5-fold. We do recognize that more complex microbial communities may require more thermocycling, more variable regions, or more input DNA, but these are all factors that must be considered and balanced for the most effective implementation. An alpha diversity curve plotted against reads is very useful in determining the amount of sequencing reads needed to saturate the community diversity, and is recommended for novel community studies and experimental design.
Jefferson Lai– Applications Scientist, Labcyte, Inc., San Jose, California
San Jose, California
Jefferson attended the University of California, Berkeley, for his undergraduate. He graduated with a BS in Microbial Biology and worked in Dr. Steve Lindow's lab developing a transposon mutagenesis screen for quorum sensing in Pseudomonas syringae. After, Jefferson began working in the synthetic biology industry, first at Amyris Inc for 3 years, then Intrexon Corp for 4 years, both as Associate Scientist, before joining Labcyte Inc as an Applications Scientist. At Amyris, Jefferson worked on replacing Saccharomyces cerevisiae native central metabolism with an augmented one to achieve yields greater than theoretically possible using native metabolism. This work has been published in Nature. At Intrexon, Jefferson developed genetic tools to engineer into a non-model host and metabolic engineering for production of a chemical, then developed, operationalized, and managed the in-house NGS pipeline. At Labcyte, Jefferson develops processes used in synthetic biology using the Labcyte Echo acoustic liquid handler.