Refining liquid biopsy generating more information from cell-free DNA with duet evoC

Refining liquid biopsy: generating more information from cell free DNA with duet evoC

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  • Fabio Puddu,
  • Tom Charlesworth
  • Robert Crawford
  • Nick Harding
  • Annelie Johansson
  • Ermira Leslie
  • Casper K Lumby
  • Aurelie Modat
  • Jamie Scotcher
  • Michael S Wilson
  • Shirong Yu
  • Jens Füllgrabe
  • Walraj S Gosal
  • Páidí Creed

1. Introduction

Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease and subtype. However, a standard blood draw yields an average of only 10 ng of cfDNA, of which DNA derived from the tumour is a small minority.

Genetic and methylation data together have been shown to be more powerful for the detection of early cancer than either alone. Constrained to measuring four states of information, existing NGS-based technologies sacrifice genetic information for methylation calling.

StateStandard sequencing protocolProtocol with C→T deamination
Overview of duet multiomics solution as part of an NGS workflow

2. duet multiomics solution evoC

  1. Strand synthesis – creates a single molecule with a direct copy of the original information tethered together with a hairpin. The copy strand is without cytosine modifications initially, but importantly, utilises a high fidelity methyltransferase to copy over only 5mC from the original to the copy strand.
  2. Sequencing paired-end read-generates sequence information after protection of cytosine modifications followed by deamination of all remaining cytosines (read as thymine in NGS).
  3. Read resolution– aligns original and copy strands to correctly call all 4 canonical bases along with 5mC and 5hmC.
  4. Aligned (4 base) reads with 5mC & 5hmC are tagged (6 base information)
The duet evoC workflow for 6-base sequencing

duet multiomics solution evoC is a 6-base calling technology that reads all four canonical bases plus 5mC and 5hmC.

3. Accurate genetic and epigenetic data

Complete accurate genome and methylome information from duet evoC. Genomic information is provided in full at higher or equivalent accuracy as other genetic and epigenetic methods (Figure 3A).

SNP calling accuracy for duet evoC
Epigenetic calling accuracy for duet evoC

Simultaneously full methylome information is provided at high accuracy for both 5mC and 5hmC (Figure 3B). Figure (A) uses data generated on the Genome-in-a-bottle reference materials.

4. More information from a single cfDNA sample

Complete accurate genome and methylome information from duet evoC. Genomic information is provided in full at higher or equivalent accuracy as other genetic and epigenetic methods (Figure 3A).

Germline variants identified in CRC cfDNA samples using duet evoC
Fragment length profile of healthy and CRC stage I-IV cfDNA samples
Deviation from dinucleosomal in healthy and CRC stage I-IV cfDNA samples identified with duet evoC
IGV example of variant associated methylation identified using duet evoC
Differentially methylated regions and differentially hydroxymethylated regions in CRC cfDNA samples identified with duet evoC

cfDNA is thought to enter the bloodstream through apoptosis or necrosis, with cfDNA from healthy and cancer tissues released into the blood of cancer patients. Nucleosome complexes partially protect cfDNA from nuclease degradation which can be seen in the fragment-length profile. Lengths tend to follow mononucleosomal distributions ranging between 150-200 bp and multiples thereof, with the majority being mononucleosomal. In circulating tumour DNA (ctDNA) many fragments are ~20bp shorter than the nucleosomal profiles just described, leading to use of fragment size distributions liquid biopsy features. As well as producing genetic variant information (A), duet evoC reads can be used to infer the length of the original fragment. Fragment size data obtained with duet evoC align (B) with what has been previously observed. Copy number information can be extracted from cfDNA analysed with duet evoC data (Cdata analysed with CNVkit), as well as information about differentially methylated regions (E). Variant associated methylation can be discovered directly from the combined genetic and epigenetic information (D – G/A variant in the NDUFA9 gene which has been associated with cancer).

All figures use data generated from cell-free DNA extracted from plasma taken from a cohort of patients with colorectal cancer and healthy volunteers.

5. The promise of earlier cancer detection

5mC and 5hmC have different roles and 5hmC has been shown to be of particular importance for the biology of enhancers, with 5hmC most prevalent in primed enhancers. We conducted differential methylation analysis comparing mean 5mC and 5hmC levels at genomic features and discovered enhancers that were able to discriminate between stage IV CRC and healthy from a 10ng cfDNA sample.

A set of enhancers with statistically significant hypomethylation at stage IV demonstrated statistically significant hypermethylation in stage I CRC samples. These enhancers appear repressed in healthy individuals, primed in early stage CRC and active in stage IV CRC (B) and these observations are lost when using a one-dimensional modC readout for modified cytosines (A).

These data illustrate the potential of 6-base data for (i) better understanding tumour biology and (ii) discovery of novel features that may be used to build a classifier for early stage cancer.

A one-dimensional modC readout cannot differentiate early stage CRC from healthy cfDNA samples
The 6-base readout from duet evoC using 5mC and 5hmC can differentiate healthy, stage I and stage IV CRC cfDNA samples

6. Conclusions

We have presented data illustrating the potential of duet evoC for liquid biopsy. With duet evoC it is possible to obtain multi-modal information, including SNPs, methylation, hydroxymethylation, fragmentomics, copy number variation and novel 2 dimensional biomarkers, from a single low-input sample of cfDNA.

duet evoC, which provides 6-base sequencing, is available to pre-order now as a product from biomodal.

See these posters for more on duet evoC:

Cambridge Epigenetix is now biomodal