Scale Down of Next Generation Sequencing for QC of DNA Assemblies

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Elaine Shapland, Elaine Shapland, Jed Dean, Victor Holmes

Amyris, United States

Technologies for assembling of multi-Kilobase synthetic DNA constructs are rapidly outpacing technologies for verifying that the final molecules match design specifications to the base pair. Traditional methods of Sanger sequencing become cost prohibitive when assemblies are large. Next-generation sequencing offers the necessary cost reduction, but for state of the art workflows the throughput is too low. To address this, Amyris has undertaken development of a high-throughput next-generation sequencing pipeline for DNA assemblies that relies on the Illumina Nextera sample preparation protocol and a single MiSeq. To match the cost of current QC methods, the Nextera sample preparation reaction volume must be scaled down more than 100-fold and each sequencing reaction should include more than 500 pooled and barcoded plasmid samples. Several steps of the pipeline require significant reengineering to meet the requirements imposed by scale and cost. When operationalized, the pipeline will provide base-pair resolution and automated data analysis for the several thousand assemblies Amyris generates each month.