Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

Lorna Suckling,Ciaran Mcfarlane,Chelsea Sawyer,Stephen P Chambers,Richard I Kitney,David W Mcclymont,Paul S Freemont

doi:10.1016/j.synbio.2019.01.002

Abstract

High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.

Highlights

The rapid advancement of next-generation sequencing (NGS) technologies has revolutionised genomics research [1]
We hypothesised that genomic DNA (gDNA) contamination was preventing the transfer of the affected plasmid DNA samples on the Echo®
It became important to ensure that plasmid DNA samples, prepared using a high-throughput plasmid isolation method [16], were free from gDNA contamination, prior to their transfer on the Echo®

Summary

Introduction

The rapid advancement of next-generation sequencing (NGS) technologies has revolutionised genomics research [1]. In order to reduce the cost of sequencing large numbers of samples simultaneously, methods have been developed for barcoding and miniaturising the preparation of libraries for NGS [2,3,4]. We describe the development of a method for the high-throughput preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. This method prepares libraries directly from purified plasmid DNA, without the use of rolling circle amplification (RCA), as has been described previously [3]. DNA assembly methods, such as golden gate [8], bring multiple

Methods

Results

Discussion

Conclusion