Sequencing library preparation for MinIONTM and PromethIONTM

Several gDNA library-prep protocols are available, offering low DNA input, rapid reaction and ultra-long reads

Fig. 1 Schematic representation of ligation-based 2D library preparation

We have developed workflows to maximise throughput from both high- and low-input samples, without the need for PCR (Figs. 1a and 1b). Omitting the PCR step means that amplification bias is eliminated and several hours of laboratory time are saved. The PCR-free low-input protocol is compatible with longer fragments than the PCR-based protocol, giving the user longer reads. Using the low-input protocol, the user loads the library into the MinION while strands are still bound to the beads. Gravity draws the beads onto the membrane surface, increasing the local concentration of DNA molecules on the membrane.

For applications where the accuracy of a 1D read (template strand only) is of sufficient quality for the downstream analysis, we have developed a rapid library prep. This prep uses a transposase enzyme, which simultaneously fragments and attaches adapters to double-stranded DNA. Starting with a DNA extract, the entire library prep can be performed in approximately 5 minutes (Fig. 2). In spite of the obligatory fragmentation step, this library prep is capable of generating extremely long reads. We are currently developing a 2D version of this library prep, which will take approximately 30 minutes to get from gDNA to the finished library.

Although all of our gDNA library preparations are compatible with reads of several kilobases in length, there are some applications for which it would be preferable to have even longer reads, such as de novo assembly of complex genomes, or phasing of SNPs over long distances. To address this need, we have developed a workflow which is capable of depleting shorter fragments from a library, leaving only the long fragments (Fig. 3). It is necessary to begin the library prep with high molecular weight DNA and to take precautions to avoid shearing the strands during preparation.

In this workflow the library is prepared using any of the PCR-free protocols, being careful not to shear the DNA, and the adapter-ligated library is then size-selected on a BluePippin. Figs. 4a and 4b show a library prepared using the standard ligation-based 2D protocol before and after size selection. Depending on the fragment size in the original gDNA sample, this protocol is capable of generating extremely long reads. Fig. 4c shows a read, obtained from an E. coli gDNA extract, in which the template and complement strands are each 255 kb, giving an overall sequence length of 510 kb.

Nanopore sequencing 2D library prep and low-input protocol

Rapid 1D and 2D library preps

Ultra-long reads using Blue Pippin size selection

Size selection removes fragments below a specified length

Fig. 3 Ultra-long-read workflow

Fig. 2 Schematic representation of rapid 1D and 2D protocols

Fig. 4 Ultra-long reads from the Blue Pippin workflow

Contact: info@nanoporetech.com More information at: www.nanoporetech.com and publications.nanoporetech.com

© 2016 Oxford Nanopore Technologies. All rights reserved.Version 1

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