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