Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries

Abstract

Synthetic biology utilises the Design-Build-Test-Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilise this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using PCR is introduced and sequencing data is analysed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 x 96 well plates) were processed in a single workflow in 72 hours (from E. coli colonies to analysed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, SNP analysis and gene synthesis.

Efficient multiplexed gene regulation inSaccharomyces cerevisiaeusing dCas12a

“…The combination of these two approaches for strain construction reduced the total construction time by omitting cloning steps and sequence verification of individual samples. A set of unique barcodes was used to generate a sequencing template allowing the resulting PCR amplicons to be pooled and analyzed in a single sequencing run (similar to the approach in ( 49 )).…”

Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

“…Cloning used a standard laboratory strain of E. coli, and bacterial cells for highefficiency transformation were prepared in-house without specialized equipment. High-throughput sequencing is predicted to soon become the routine verification method for synthetic DNA constructs (Shapland et al 2015;Currin et al 2019;Gallegos et al 2020). The full-length sequence of the plasmid assembled was verified using the Illumina platform in a single sequencing reaction that required no custom primers or data analysis pipelines, and avoided time-consuming primer walking strategies.…”

Combinatorial metabolic pathway assembly approaches and toolkits for modular assembly

“…Long-read NGS sequencing is also employed: D'Amore et al ( 2017) developed a high-throughput SMRT (Single Molecule, Real-Time) Gate method to validate plasmid inserts using Golden Gate and Pacific Biosciences technology. In addition, a new PCR barcoding system and analysis algorithm were recently described that facilitate fast high-throughput plasmid sequencing using Oxford Nanopore technology (Currin et al, 2019).…”