GRAIL, LLC

Compositions and methods are provided for amplifying nucleic acids, including cell free nucleic acid fragments, in preparation for sequencing. Methods are provided for making circularized nucleic acid templates having the structure [T]-[PS1]-[L]-[PS2] or [PS1]-[L]-[PS2]-[T′], where (a) T is a target nucleic acid and T′ is a complement to a target nucleic acid; (b) each of PS1 and PS2 is a nucleic acid primer site; (c) L is a linker having a primer extension reaction terminating organic molecule; and the structure is circularized by binding a 5′ end thereof to a 3′ end thereof. Target sequences in the circularized templates are amplified by binding to PS1 a primer complimentary to PS1 and binding to PS2 a primer complimentary to PS2 and copying the target sequences by a primer extension reaction. Advantages include a reduction in ligation steps, which can result in fewer clean up steps and improved library conversion efficiency.

Gene copy number variations are identified for genes in a targeted gene panel. For each gene, coverage at each base position across the gene is determined. The coverage at each base position can be influenced by the hybridization probes that are used to determine the base level coverage of the base position. The base level coverage for each base position is normalized to account for the characteristics of the hybridization probes. To determine whether a copy number variation exists for a gene, the base level coverage of base positions across the gene for a subject is analyzed to determine whether it deviates from the base level coverage of base positions across the gene for previously analyzed, healthy individuals. If a significant deviation exists, a copy number variation for the gene is called.

In various aspects, the present disclosure provides methods, compositions, reactions mixtures, kits, and systems for analysis of cell-free nucleic acid molecules (e.g., cfRNA and/or cfDNA) from a urine sample. In some embodiments, the analysis is an analysis of methylation patterns in target genomic regions among cfDNA fragments in a urine sample. In some embodiments, compositions include a plurality of different bait oligonucleotides. Methods for the detection of cancer of various cancer types are also provided.

Aspects of the invention relate to methods for preparing and analyzing a sequencing library from a mixed cell-free DNA (cfDNA) sample, wherein the mixed sample includes double-stranded DNA (dsDNA), damaged dsDNA (e.g., nicked dsDNA), and single-stranded DNA (ssDNA) molecules. The subject methods facilitate the collection of information from dsDNA, ssDNA and damaged DNA (e.g., nicked DNA) molecules in a sample, thereby providing enhanced diagnostic information as compared to sequencing libraries that are prepared from dsDNA alone.

A method of processing an input sample, as well as related kits and compositions, is provided herein. In various instances, the disclosure relates to providing an input sample comprising nucleic acid fragments, wherein in at least a portion of the nucleic acid fragments each fragment comprises one or more methylated cytosines; converting unmethylated cytosines of nucleic acid fragments of the input sample to uracils, yielding converted fragments; copying the converted fragments using a mixture of nucleotides, the mixture comprising a mixture of: binding moiety-modified cytosines and binding moiety-lacking cytosines; binding moiety-modified guanines and binding moiety-lacking guanines; or binding moiety-modified cytosines, binding moiety-lacking cytosines, binding moiety-modified guanines, and binding moiety-lacking guanines; wherein the copying yields a mixture of binding moiety-modified fragments and unmodified fragments which may be separated to provide a set of fragments enriched for hypermethylated fragments.

Methods for preparing enriched sequencing libraries from test samples that contain double-stranded deoxyribonucleic acid (dsDNA) are provided.

Methods for preparing enriched sequencing libraries from test samples that contain double-stranded deoxyribonucleic acid (dsDNA) are provided.

Methods for generating a sequencing library from a sample comprising a plurality of single-stranded DNA molecules are provided, along with methods of using the generated sequencing library for detecting cancer, determining cancer stage, monitoring cancer progression, and/or determining a cancer classification from a test sample obtained from a subject.

Provided herein are methods for enriching a biological sample for a target nucleic acid, and analyzing the nucleic acid. In some cases, a biological sample is enriched for target nucleic acids associated with a cancer or tumor. In some cases, a biological sample is enriched for target nucleic acids, and the target nucleic acids vary in length. In some cases, one or more probes are used to enrich the biological sample for the target nucleic acid. In some cases, one or more probes hybridize to one or more ends of a target nucleic acid.

Aspects of the invention include methods for preparing sequencing libraries, performing sequencing procedures that can correct for process-related errors, and identifying rare variants that are or may be indicative of cancer.