High-throughput automated microfluidic sample preparation for accurate microbial genomics

Abstract

Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications.

Increasing antibiotic susceptibility inStaphylococcus aureusin Boston, Massachusetts, 2000-2014: an observational study

“…We collected non-duplicate clinical specimens from patients ≥18 years of age who had specimens submitted to the BWH clinical microbiology laboratory between January 1, a microfluidic platform using the Illumina Nextera protocol as described 32 and sequenced on an Illumina platform. A detailed description of isolate preparation is located in the eMethods section of the supplement.…”

Multiplexed enrichment and genomic profiling of peripheral immune cell subsets on a microfluidic chip

“…This system integrates microfluidic liquid handling with magnetic affinity purification. The device is fabricated using established methods for two-layer soft-lithography 24 and contains 39 micromechanical valves controlled by an external pneumatic valve controller 25 . The device consists of three main chambers partitioned by microvalves ( Fig.…”

“…The microfluidic device was fabricated using a previously published protocol 25 with minor modifications. Flow layer molds were patterned in 3 steps: (1) rectangular 75 µm, (2) rectangular 200 µm, and (3) rounded 60 µm.…”

Continuous Microfluidic Purification of DNA Using Magnetophoresis

“…Purification and isolation of nucleotide samples like deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) is an important step for subsequent characterization and application of various biochemical reaction [1][2][3]. The common methods of purification of DNA include electrophoresis [4], high salt precipitation [5], spin column [6], and magnetic beads [7], etc.…”