Oxford nanopore minion

London calling!

26th-27th may 2016

Burn-in experiment

Other experiments

https://metrichor.com/workflow_instance/80467

We ran 2 more experiments: Whole phage genome and long range PCR amplicon (approx. 12kb long)

These experiments weren't even as successful as lambda burn-in, mainly because expiration of the flowcell

More details will be presented by Nik

Enter the Metrichor!

• Cloud based platform for realtime analysis of sequenced data
• offers a downloadable client that connects the instrument to analytic platform
• motto of the company-"internet of living things"

From signal to basecall

Areas of application

This is called "squigglespace"

reads quality:

-1Directional (template)

-1D (complement)

-2D

-Pass (Q score>9)

-Fail

Metrichor

each pentamer characterized by discrete value. 1024 theoretical unique current values (4^5)

• Reads are generated in FAST5 format - HDF file
• 1 file is created from each read
• Bioinformatic tools are required to generate .fastq files

Fast - "realtime" applications, disease epidemics, monitoring (smuggling of goods). competition: RE fingerprinting, rapid PCR methods

Field - work in open terrain, ecology, lab-in-a-van, minimalisation of power-consuming equipment, isothermal amplification. Competition?

Long - for de novo sequencing, needs careful sample handling, record to date is around 200kb. Competition = PacBio sequencers

Lots - getting the maximum output for the best price. Not the strongest suit of minION, competing with Illumina sequencing

Pricing

ILMN Miseq: approx. 33000,- CZK/300v2 run, output 6Gb... 5500,- per Gb

When considering specified 6Gb/48h run: 16556/6 = 2760,- per Gb

Considering the hall of fame data - around 15000 czk per Gb is more realistic

Scipts!

Python libraries that control the parameters of the sequencing run:

• voltage on membrane
• re-mux scans
• re-mux frequency

Scripts can be customised and the output can be improved very much!

http://porecamp.github.io/pdf/Porecamp_RunningMinION_JT.pdf

Publications

"Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool"

Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions. We successfully used a novel, pocket-sized nanopore sequencer at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak.

Bacterial resistance genes identification

...Here, we address these challenges, using a de Bruijn graph comparison of clinical isolate and curated knowledge-base to identify species and predict resistance profile, including minor populations. This is implemented in a package, Mykrobe predictor, for S. aureus and M. tuberculosis, running in under three minutes on a laptop from raw data. For S. aureus, we train and validate in 495/471 samples respectively, finding error rates comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.3%/99.5% across 12 drugs. For M. tuberculosis, we identify species and predict resistance with specificity of 98.5% (training/validating on 1920/1609 samples). Sensitivity of 82.6% is limited by current understanding of genetic mechanisms. We also show that analysis of minor populations increases power to detect phenotypic resistance in second-line drugs without appreciable loss of specificity. Finally, we demonstrate feasibility of an emerging single-molecule sequencing technique.

Introducing...

Snake venom gland cDNA sequencing using the Oxford Nanopore MinION portable DNA sequencer

...Here we show that single-molecule cDNA sequencing using the MinION accurately characterises venom toxin-encoding genes in the painted saw-scaled viper, Echis coloratus. We find the raw sequencing error rate to be around 12%, improved to 0-2% with hybrid error correction and 3% with de novo error correction. Our corrected data provides full coding sequences and 5′ and 3′ UTRs for 29 of 33 candidate venom toxins detected, far superior to Illumina data (13/40 complete) and Sanger-based ESTs (15/29). We suggest that, should the current pace of improvement continue, the MinION will become the default approach for cDNA sequencing in a variety of species.

Types of libraries

Determining Exon Connectivity in Complex mRNAs by Nanopore Sequencing

Though powerful, short-read high throughput RNA sequencing is limited in its ability to directly measure exon connectivity in mRNAs containing multiple alternative exons located farther apart than the maximum read lengths. Here, we use the Oxford Nanopore MinION™ sequencer to identify 7,899 ‘full-length’ isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be an important method for comprehensive transcriptome characterization.

MAP - Minion access programme

Nanopore sequencing detects structural variants in cancer

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300bp) of 2nd generation sequencing..... We propose to use the long-reads (up to 20kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. ... its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency. We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.

already developed:

genomic DNA

Amplicon sequencing

Low input gDNA

native/PCR barcoding

cDNA libraries (strand switching protocol)

in development/experimental:

direct RNA seq

protein analysis

DNA modfications (methyl-seq)

MinION nanopore sequencing of an influenza genome

Influenza epidemics and pandemics have significant impacts on economies, morbidity and mortality worldwide. The ability to rapidly, and accurately, sequence influenza viruses is instrumental in the prevention and mitigation of influenza. All eight influenza genes from an influenza A viruses were amplified simultaneously and then subjected to sequencing on a MinION nanopore sequencer. A complete influenza virus genome was obtained that shared greater than 99% identity with sequence data obtained from the Illumina Miseq and traditional Sanger-sequencing. The laboratory infrastructure and computing resources used to perform this experiment on the MinION nanopore sequencer would be available in most molecular laboratories around the world. Using this system, the concept of portability, and thus sequencing influenza viruses in the clinic or field is now tenable.

• ONT's way to develop the platform
• community based protocols and tools
• Burn-in experiment, FC returning to ONT
• Started in Jan 2014, our lab joined by the end of 2014

Library preparation

Alternatives and protocol modifications

variable frag size - 6-20kb

Hall of Fame

• using a fine (ga.26) needle to perform the shearing = saving 750 czk/sample, generates fragments ~25kb

• enzymatic fragmentation also possible

Flowcell recycling:

ONT supplies a wash kit, that can prepare the flowcell fore another round of sequencing

the pores will deteriorate with each run, ultimately the FC is lost

ONT's specification:

output up to 6Gb in 48h

basecall accuracy 96% - depending on analysis software

Modified bases detection

reality???

https://nanoporetech.com/applications/dna-nanopore-sequencing

• 1D libraries can be prepared in 15 minutes

• Protocol for generating ultra-long reads provided by the community

(optional) NEBNext FFPE DNA Repair Mix

• Input 1-1.5 ng gDNA
• low input module - 20-100 ng gDNA
• Non-degraded! fragments should be <30kb
• PCR free workflow
• balancing read lenght vs library yield

motor protein+hairpin protein

Followed by MyOne T1 biotinylated beads pulldown

=>flowcell loading, possible Re-loadinq each 6 hours

Nanopore Sequencing

Filip Pardy, CF genomics

CEITEC MU 3.3. 2016