Oxford NanoporeTechnologies

Hadas Keren-Shaul Advanced Sequencing Technologies (Sandbox), LSCFFeinberg June 2018

LSCF

Nanopore sequencing – Long reads

Long read sequencing technology offers simplified and less ambiguous genome assembly

“Analysis of any living thing, by any person, in any environment”

MinION – The first and only nanopore DNA sequencer

• A portable, real-time, long-read, low-cost device

• Designed to bring easy biological analyses to scientific research, education or a range of real-world applications such as disease/pathogen surveillance, environmental monitoring, food-chain surveillance, self-quantification or even microgravity biology

• Every flow cell - 10-20 Gb of DNA sequence data

MinION sequencing device

Lu et al., Review: Oxford nanopore MinION sequencing and genome assembly, Genomics Proteomics Bioinformatics, 2016

What is a Nanopore?

• A very small hole – nano-scale

• Oxford nanopore passes an ionic current through nanoporesand measures the changes as biological molecules pass through the nanopore or near it

• Holes can be created by protein puncturing membranes (biological nanopores) or in solid materials (solid-state nanopores)

How does it work?

https://nanoporetech.com/how-it-works

• A strand of DNA is passed through a nanopore

• The current is changed as the bases G, A, T and C pass through the pore in different combinations

Direct molecular analysis

• A direct electronic analysis of the target molecule – native analysis

• Sequencing native DNA (and RNA) avoids bias (no amplification)

Rapid sequencing kit

• Input – extracted gDNA

• Time – 10 minutes

• A transposase which simultaneously cleaves template molecules and attaches tags to the cleaved ends

cDNA PCR sequencing kit

• Input – 50 ng poly(A) RNA

• RT and PCR

Direct RNA sequencing kit

• Input – 500 ng poly(A) RNA

• No RT or PCR bias

• Useful for transcripts that are difficult to reverse transcribe

The Nanopore sequencing workflow

• Real Time data: no fixed run time allows on-demand sequencing and real time adjustment

Why MinION?

Applications

Making nanopore scalable

A core sensing unit of a nanopore set in an arrayed sensor chip

PacBio vs. Oxford Nanopore

• PacBio – Sequence by synthesis

• Nanopore – monitoring the transit of a DNA molecule through a hole and measuring variations in electric current

PacBio vs. Oxford Nanopore

• PacBio has overall better data quality

• ONT provides higher yield per flow cell

Thank you for listening