Thinking beyond library construction

Contributed by Carol Huang

We have recently introduced a newer version of the Nanopore sequencing device, MinION MK 1B into the laboratory. It’s a portable, real-time sequencing device.

The Nanopore MinION sequencing platform has great advanced features. Upon reach objectives, it can generate 10 to 20 GB DNA sequencing data from each flow cell. With different fragmentation options ultra-long reads, hundreds of kb in length, were made possible. Since I have been engaged in NGS library construction for many years, the more impressive feature of this is the time saved on sequencing library preparation in comparison with other platforms. Sequencing libraries can be made in less than an hour using the library kit we picked, while to make long-read library could take 2 days by using other sequencing platforms. We were able to pick the sequencing data set from the first five minutes of sequencing run and identified the serotype of the microbe tested. These features will be very practical in the field of pathogen infection control.

One of the key factors to ensure high-quality data outcome is the quality of input materials, RNA, DNA, their integrity, and purity. Purity, 260/280 and 260/230 ratios, affects enzyme efficiency during library preparation, organic residuals would interfere with chemical reaction and might damage nanopore membranes. There are 512 nanopore channels available to capture library fragments. Small fragments always get captured, occupy nanopore channels first, which would prevent those bigger fragments from being sequenced and furthermore reduces sequencing efficiency. So for the best results, we need to have high purity input DNA /RNA and keep narrow final library size distribution, eliminate outsized final library fragments, small size in particular.

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