PUBLICATIONS

featuring the Echo acoustic technology

111 Total Publications

TITLES and AUTHORS

  • Year
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  • + Abstract
  • High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
  • Jeremiah J. Minich, et al.
  • Institution: Marine Biology Research Division, Scripps Institution of Oceanography, UCSD
  • Publication: American Society for Microbiology
  • 2018

Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation 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. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements.

  • Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation
  • Madeline Y Mayday, et al.
  • Institution: University of California San Francisco
  • Publication: bioRxiv
  • 2018

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

  • Miniaturization and optimization of 384-well compatible metagenomic sequencing library preparation
  • Madeline Y Mayday, et al.
  • Institution: UCSF School of Medicine, Chan Zuckerberg Biohub
  • Publication: bioRxiv
  • 2018

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next-generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved a savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

  • Rapid Prototyping Platform for Saccharomyces cerevisiae Using Computer-Aided Genetic Design Enabled by Parallel Software and Workcell Platform Development
  • P.D. Rajakumar, et al.
  • Institution: The London DNA Foundry
  • Publication: SAGE Journals - SLAS Technology
  • 2018
  •  

Biofoundries have enabled the ability to automate the construction of genetic constructs using computer-aided design. In this study, we have developed the methodology required to abstract and automate the construction of yeast-compatible designs. We demonstrate the use of our in-house software tool, AMOS, to coordinate with design software, JMP, and robotic liquid handling platforms to successfully manage the construction of a library of 88 yeast expression plasmids. In this proof-of-principle study, we used three fluorescent genes as proxy for three enzyme coding sequences. Our platform has been designed to quickly iterate around a design cycle of four protein coding sequences per plasmid, with larger numbers possible with multiplexed genome integrations in Saccharomyces cerevisiae. This work highlights how developing scalable new biotechnology applications requires a close integration between software development, liquid handling robotics, and protocol development.

  • Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria
  • Simon J. Moore, et al.
  • Institution: Centre for Synthetic Biology and Innovation, Imperial College London
  • Publication: PNAS
  • 2018
  •  

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.

  • Publication / Type:PNAS
  • Related Subject:bacteria research, txtl, bayesian, synthetic biology
  • Identifying inhibitors of the Leishmania inositol phosphorylceramide synthase with antiprotozoal activity using a yeast-based assay and ultra-high throughput screening platform
  • Jennifer L. Norcliffe, et al.
  • Institution: Department of Biosciences, Durham University
  • Publication: Nature
  • 2018
  •  

Leishmaniasis is a Neglected Tropical Disease caused by the insect-vector borne protozoan parasite, Leishmania species. Infection affects millions of the world’s poorest, however vaccines are absent and drug therapy limited. Recently, public-private partnerships have developed to identify new modes of controlling leishmaniasis. Drug discovery is a significant part of these efforts and here we describe the development and utilization of a novel assay to identify antiprotozoal inhibitors of the Leishmania enzyme, inositol phosphorylceramide (IPC) synthase. IPC synthase is a membrane-bound protein with multiple transmembrane domains, meaning that a conventional in vitro assay using purified protein in solution is highly challenging. Therefore, we utilized Saccharomyces cerevisiae as a vehicle to facilitate ultra-high throughput screening of 1.8 million compounds. Antileishmanial benzazepanes were identified and shown to inhibit the enzyme at nanomolar concentrations. Further chemistry produced a benzazepane that demonstrated potent and specific inhibition of IPC synthase in the Leishmania cell.

  • Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format
  • Leonardo B. Pinheiro, et. al.
  • Institution: National Measurement Institute (NMI), Sydney, Australia, et al.
  • Publication: Analytical Chemistry
  • 2017
  •  

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

  • Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond
  • Leslie A. Mitchell, et. al.
  • Institution: NYU School of Medicine, Boeke Lab
  • Publication: Science
  • 2017

Total synthesis of designer chromosomes and genomes is a new paradigm for the study of genetics and biological systems. The Sc2.0 project is building a designer yeast genome from scratch to test and extend the lim- its of our biological knowledge. Here we describe the design, rapid assembly, and characterization of synthetic chromosome VI (synVI). Further, we investigate the phenotypic, transcriptomic, and some consolidation to uncover possible synthetic genetic interactions and/or perturbations of native cellular networks as the number of designer changes increases is the next major step for the Sc2.0 project.

  • Bug mapping and fitness testing of chemically synthesized chromosome X
  • Yi Wu, et. al.
  • Institution: NYU School of Medicine, Boeke Lab
  • Publication: Science
  • 2017

Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity, com- prehensiveness, and accuracy of the knowl- edge amassed by the yeast community to date. The international synthetic yeast genome project (Sc2.0) aims to build an entirely de- signer, synthetic Saccharomyces cerevisiae ge- nome. The synthetic genome is designed to increase genome stability and genetic flexi- bility while maintaining cell fitness near that of the wild type. A major challenge for a ge- nome synthesis lies in identifying and elim- inating fitness-reducing sequence variants referred to as “bugs.”

  • Engineering DNA nanotubes for resilience in an E. coli TXTL system
  • Melissa A. Klocke, et.al.
  • Institution: Mechanical Engineering, University of California, Riverside
  • Publication: Synthetic Biology
  • 2017
  •  

Deoxyribonucleic acid (DNA) nanotechnology is a growing field with potential intracellular applications. In this work, we use an Escherichia coli cell-free transcription–translation (TXTL) system to assay the robustness of DNA nanotubes in a cytoplasmic environment. TXTL recapitulates physiological conditions as well as strong linear DNA degradation through the RecBCD complex, the major exonuclease in E. coli. We demonstrate that chemical modifications of the tiles making up DNA nanotubes extend their viability in TXTL for more than 24 h, with phosphorothioation of the sticky end backbone being the most effective. Furthermore, we show that a Chi-site double-stranded DNA, an inhibitor of the RecBCD complex, extends DNA nanotube lifetime significantly. These complementary approaches are a first step toward a systematic prototyping of DNA nanostructures in active cell-free cytoplasmic environments and expand the scope of TXTL utilization for bioengineering.

  • Publication / Type:Synthetic Biology
  • Related Subject:Echo 525, Synthetic Biology, DNA