Methods for multi-resolution analysis of cell-free nucleic acids

Inventors

Chudova, Darya • Eltoukhy, Helmy • MORTIMER, Stefanie Ann Ward • Abdueva, Diana

Assignees

Guardant Health Inc

Abstract

The present disclosure provides a method for enriching for multiple genomic regions using a first bait set that selectively hybridizes to a first set of genomic regions of a nucleic acid sample and a second bait set that selectively hybridizes to a second set of genomic regions of the nucleic acid sample. These bait set panels can selectively enrich for one or more nucleosome-associated regions of a genome, said nucleosome-associated regions comprising genomic regions having one or more genomic base positions with differential nucleosomal occupancy, wherein the differential nucleosomal occupancy is characteristic of a cell or tissue type of origin or disease state.

Core Innovation

The invention provides a method for multi-resolution analysis of cell-free DNA (cfDNA). The method includes providing a sample comprising cfDNA and enriching cfDNA, or amplification products thereof, from hotspot regions and backbone regions using a bait set panel. The bait set panel preferentially enriches the hotspot regions compared to the backbone regions, where the backbone regions are larger genomic regions compared to the hotspot regions.

After enrichment, the method sequences the enriched set of nucleic acids, or amplification products thereof. The enrichment produces higher average read depth for the hotspot regions compared to the average read depth for the backbone regions. The multi-resolution aspect partitions genomic analysis into hotspot regions and larger backbone regions, enabling differential capture and read depth across resolutions.

The document further describes multi-resolution cfDNA analysis in terms of nucleosome-associated genomic regions and differential nucleosomal occupancy. Nucleosome-associated regions are defined by differential nucleosomal occupancy characteristic of a cell/tissue type of origin or disease state, and the bait panels include multi-bait schemes for enriching hotspot genomic regions versus backbone genomic regions with saturation-controlled probe concentrations.

In addition to enrichment and sequencing, the document describes improving indel-calling accuracy using hypothesis testing with model parameters to classify candidate indels as true indels versus introduced indels. The approach accounts for sequencing and amplification-related errors, and it references analytical performance improvements assessed using hotspot/backbone titration experiments and analytical sensitivity/specificity/PPV metrics.

Claims Coverage

The identified independent claim is directed to a multi-resolution cfDNA analysis method using hotspot versus backbone enrichment with a bait set panel, followed by sequencing. The claim set further includes inventive features for target-specific oligonucleotides, saturation-controlled capture, read-depth constraints, variant detection with comparative sensitivity, and an alternative nucleosome-associated enrichment basis.

The coverage centers on preferential bait-based enrichment of hotspot regions versus larger backbone regions to achieve differential read depth, followed by sequencing. The dependent features further specify target-specific oligonucleotides, saturation-controlled hotspot versus backbone capture, sequencing read-depth constraints, comparative sensitivity for genetic variant detection, and enrichment based on nucleosome-associated regions defined by differential nucleosomal occupancy.

Stated Advantages

Documented Applications