Nanopore sequencing for intraoperative cancer diagnosis

Outline first, second and third generation sequencing

First generation sequencing is sanger sequencing, discovered in 1970, where chain termination occurs using fluorescent dideoxynucleotides.

Second generation sequencing (next generation sequencing) is illumina, sequencing by synthesis using florescent labelled dNTPs in amplification (PCR)

Third generation sequencing (long-read) also known as oxford nanopore technologies, this can sequence native DNA/RNA straight from the cell, it passes through a nanopore and changes in current correspond to base identity and is recorded.

What are the key features and applications of oxford nanopore technology?

ONT allows long reads, up to 4Mb of DNA,. it allows real time output enabling targeted and adaptive sequencing which can directly connect to computers to read. It can work on native DNA straight from the cell to detect epigenetic modifications such as methylation. It is portable, rapid and scalable.

The main application of ONT is scalable sequencing for diagnostic use

What are the challenges of CNS tumours?

CNS tumours are the leading cause of cancer death in under 40s, there are ~12,000 new diagnoses per year in the UK. There is only 15% 5-year survival for common brain tumours. These mean that accurate and timely classification and diagnosis is essential and central to treatment and research. 

Why does the classification of CNS tumours matter?

1. tumours that look the same can behave differently

2. tumours that look different can be the same

3. many tumours lack distinctive morphological features

These mean that normal histology will not work optimally, and there is a need for molecular profiling.

What is adaptive sampling in cancer profiling with nanopore?

A technique that selectively enriches or depletes specific DNA regions during a run by using real-time basecalling and alignment. It matches sequences only to a target genome for a specific diagnosis. When non-target DNA is read it rejects it and looks again for target DNA.

What are epigenetic modifications that nanopore sequencing can detect?

Single nucleotide variants

Gene fusions

Promoter methylation

Copy number changes

Global methylation

Describe the current NHS diagnostic pathways and limitations of it

The current NHS diagnostic pathway requires a multidisciplinary team, and is only based in several locations around the UK. There is a long turn around time (about a month). 

Limitations include the inability to acheive a diagnosis, the grade of tumour can change during the month period, MDTs may be inneficient, oncological and surgical decisions need to be made before the months period is over,

What is ROBIN

Rapid Oxford nanopore Brain Intraoperative Nanopore classification. This is a ultrafast sequencing, can determine methylation classification within two hours, and final molecular data is available the next day.

Give a case study on the importance of intraoperative classification

• 11 month old with primitive tumour

• subtotal resection while awaiting diagnosis, later confirmed to be ependymoma.

• with rapid sequencing we could avoid a second surgery

Give a case study of real time diagnosis

3 year old with posterior fossa tumour, real time adaptive sampling diagnosed her with medulloblastoma which was confirmed intraoperatively.

What are the unique aspects of ROBIN?

• graphical interface for visualisation

• multiple classifiers for confidence

• PromethION data for high throughput

• ultra-fast library preparation

HOWEVER not licensed for diagnostic use