Nanopore sequencing from liquid biopsy: analysis of copy number variations from cell-free DNA of lung cancer patients

Filippo Martignano,Iacopo Petrini,Silvestro G Conticello,Alberto Magi,Roberto Semeraro,Alessandra Mingrino,Marzia Del Re,Stefania Crucitta,Uday Munagala

doi:10.1186/s12943-021-01327-5

Abstract

In the “precision oncology” era the characterization of tumor genetic features is a pivotal step in cancer patients’ management. Liquid biopsy approaches, such as analysis of cell-free DNA from plasma, represent a powerful and noninvasive strategy to obtain information about the genomic status of the tumor. Sequencing-based analyses of cell-free DNA, currently performed with second generation sequencers, are extremely powerful but poorly scalable and not always accessible also due to instrumentation costs. Third generation sequencing platforms, such as Nanopore sequencers, aim at overcoming these obstacles but, unfortunately, are not designed for cell-free DNA analysis.Here we present a customized workflow to exploit low-coverage Nanopore sequencing for the detection of copy number variations from plasma of cancer patients. Whole genome molecular karyotypes of 6 lung cancer patients and 4 healthy subjects were successfully produced with as few as 2 million reads, and common lung-related copy number alterations were readily detected.This is the first successful use of Nanopore sequencing for copy number profiling from plasma DNA. In this context, Nanopore represents a reliable alternative to Illumina sequencing, with the advantages of minute instrumentation costs and extremely short analysis time.The availability of protocols for Nanopore-based cell-free DNA analysis will make this analysis finally accessible, exploiting the full potential of liquid biopsy both for research and clinical purposes.

Highlights

Copy number variations (CNVs) are one of the characterizing features in many cancers: specific CNVs can define type and progression of the tumor, and are tightly linked to the diagnostic and prognostic process [1]
The most common approach is the analysis of cell-free DNA from plasma, which can be collected at different time-points to follow tumor evolution, with limited harm and risks for the patient [2, 3]
Third generation sequencing approaches, such as Nanopore technology, interrogate single molecules of DNA and produce sequences much longer than those generated by second generation sequencing (SGS) methods

Summary

Introduction

Copy number variations (CNVs) are one of the characterizing features in many cancers: specific CNVs can define type and progression of the tumor, and are tightly linked to the diagnostic and prognostic process [1]. As Nanopore technology is optimized for long read sequencing, its protocols are not ideal for analysis of short cfDNA fragments. To further confirm that these CNVs were artifacts, we sequenced the genomic DNA from white blood cells of one healthy control (HM1) and no CNVs were detected (Additional file 3: Fig. S4).

Results

Conclusion