Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform

Abstract

Real-time sequencing of short DNA reads has a wide variety of clinical and research applications including screening for mutations, target sequences and aneuploidy. We recently demonstrated that MinION, a nanopore-based DNA sequencing device the size of a USB drive, could be used for short-read DNA sequencing. In this study, an ultra-rapid multiplex library preparation and sequencing method for the MinION is presented and applied to accurately test normal diploid and aneuploidy samples' genomic DNA in under three hours, including library preparation and sequencing. This novel method shows great promise as a clinical diagnostic test for applications requiring rapid short-read DNA sequencing.

Keywords: MinION nanopore sequencing; aneuploidy detection; multiplex; small DNA; ultra-fast.

Figure 1

Comparison of MinION library preparation workflows. 2D library is a previously reported workflow (wei and williams 2016); 1D multiplex library is manufacturer’s workflow using a native barcoding kit and a 1D genomic sequencing kit on the current MinION platform; Rapid 1D multiplex library is a new rapid barcoding MinION library preparation workflow reported in this study developed to sequence short reads (<1000 bp) on the current platform. The length of each bar indicates the time needed. The steps are color-coded (Yellow: fragmentation; Red: end preparation including end-repair and dA-tail; blue: size selection and purification; dark blue: MyOne C1 bead purification; purple: sequencing).

Figure 2

Optimization of MinION library preparation. A). Optimization of ligation condition for TA ligation and 6-bp sticky-end ligation. Condition 1. The manufacturer’s suggested condition; 2. the condition reported before (wei and williams 2016); 3-5. the conditions with addition of 6%, 9%, and 12% enhancer mix. Efficiencies of 6-bp ligation were estimated using a pair of adaptor MP1-6bp and ME-6bp carrying complementary 6-bp sticky ends. Efficiencies of TA ligation were estimated using a pair of adaptor MP1-T and ME-A carrying complementary 3′T and 3′A overhangs. B). Titration experiment of Native Barcode (NB) adapter. 6.5ng, 9.8ng, 13ng of NB adapters were added in to the 1-step ligation reaction which contains the same amount of dA-tailed DNA and MP1-6bp adapter. The expected final products with 2-end ligated to a barcode and MP1-6bp adapter were marked in bold. The products separated on gel were also illustrated in cartoons (MP1-6bp adapter: green; NB adapter: blue; dA-tailed DNA: purple). C). Optimization of end-repair/dA-tailling condition. Lane 1, the input 434bp control fragment; lane 2, manufacturer’s recommended protocol; lane 3. the optimized condition; lane 4. The optimized condition with supplementation of Bst 2.0 WarmStart Polymerase. The expected products with 2-end ligated to an adapter were marked in bold and the products separated on gel were also illustrated in cartoons (434bp dA-tailed DNA: purple; MP1-T adapter: dark green). D). Optimization of AMPure XP bead purification by changing the volume of bead. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to onefold, 0.65-fold, 0.sixfold, 0.55-fold AMPure XP bead purification. The expected products are bands >500 bp, and it’s marked in bold E). Optimization of AMPure XP bead purification by adjusting the concentration of PEG in wash buffer. 100 ng 50bp ladder and 2 pmole 204bp control fragment were used as input, and subjected to 0.62-fold AMPure XP bead purification using wash buffer containing 10%, 9%, 8.5% and 8% PEG. The expected products are bands >500 bp, and it’s marked in bold. F). Optimization of tethering condition. Lane 1-5: 1µL BAM adapter with 0-4µL ELB buffer after 3min incubation at 37°C. The expected tethered BAM adapter was marked in bold. The products separated on gel were illustrated in cartoons (BAM adapter: gray; tether: pink-black).

Figure 3

MinION Run performance and assay results. A). The run performance of the rapid 1D barcoding MinION library preparation method. B). Illustation of MinION assay results for a normal male, normal female, trisomy 21 and a monosomy X. Normal and aneuploidy on each chromosome was indicated by color (Normal: black; Aneuploidy: red).