Abstract: Provided herein is method for, among other things, estimating the number of sequence variations in a sample of DNA. In some embodiments, the method can be used to estimate the mutational load of a sample. In some embodiments, the method makes use of a set of primers that have 3? ends that specifically hybridizes to a sequence that is repeated multiple times in the genome. Thermocycling a reaction mix containing the primers may produce a reaction product comprising at least 50 amplicons having a total length of at least 100 kb. This product can be sequenced to provide an estimate of the number of sequence variations in the sample, and thus the mutational load of the sample.

Abstract: Methods and apparatus for assessing quality of a genetic variant for inclusion as a biomarker in a circulating tumour DNA (ctDNA) assay. The method includes receiving genetic variants associated with a sample collected from a patient, the set of genetic variants including a first genetic variant, determining values for a plurality of characteristics associated with the first genetic variant, providing the values for the plurality of characteristics as input to a trained machine learning (ML) model, the trained ML model being trained to output a quality of a genetic variant, the quality of the genetic variant representing a likelihood that the genetic variant is both somatic and will sufficiently amplify using amplicon sequencing, and including the first genetic variant in a panel of genetic variants for use in a ctDNA assay for the patient based on the quality of the first genetic variant output from the trained ML model.

Abstract: Provided herein, among other things, is a method of treating a cancer patient without the need for a tissue biopsy. In some embodiments, the method may comprise (a) performing or having performed a sequencing assay on cell-free DNA (cfDNA) from a sample of blood from the patient to determine if the cell-free DNA comprises actionable and/or non-actionable sequence variations in one or more target genes, and (b) treating the patient using the following method: i. administering a therapy that is targeted to an actionable sequence variation if the patient is identified as having the actionable sequence variation, and ii. administering a non-targeted therapy in the absence of any follow-up genetic testing on DNA extracted from a tissue biopsy if one or more non-actionable sequence variations and no actionable sequence variations are identified.

Abstract: A method for selective sequencing of a plurality of target regions from a patient is provided. A pool of oligonucleotides comprises a first sub-population targeting the plurality of target regions and a second sub-population. Each member of the first sub-population of oligonucleotides comprises a first sequence that is complementary to one of the plurality of target regions from the patient and an identifier sequence specific to the patient, and each member of the second sub-population of oligonucleotides comprises a first sequence that is complementary to one of the plurality of target regions from a second patient and an identifier sequence specific to the second patient. A test sample from the patient is contacted with the pool, and then contacted with oligonucleotides comprising a sequence that is complementary to the identifier sequence specific to the first patient. The plurality of target regions from the patient are then selected and sequenced.

Abstract: Described herein, among other things, is a method of sequencing nucleic acids of interest (NAOIs). In some embodiments the method may comprise: providing a sample comprising NAOIs; attaching oligonucleotides to the NAOIs to provide labelled NAOIs, wherein the oligonucleotides comprise a PCR cycle counter generator sequence comprising at least one universal nucleotide base; amplifying the labelled NAOIs using PCR to provide an amplified library of NAOIs each containing a PCR cycle counter sequence; sequencing the amplified library of NAOIs to provide a set of sequence reads, wherein each sequence read comprises a NAOI-originating component and a PCR cycle counter component; and distinguishing true variants in NAOI sequences from false variants in NAOI sequences.

Abstract: The present disclosure relates to methods for detecting and targeting genomic rearrangements, in particular gene fusion events, by targeting a DNA molecule of interest with a set or pool of primers, wherein the forward primers and reverse primers produce a PCR amplification product when a genomic rearrangement is present. The present disclosure also relates to methods of bioinformatic analysis to determine whether or not the detection of an amplification product from the selective PCR is actually indicative of the presence of a gene fusion. The present disclosure also related to related methods of diagnosis and treatment of diseases and conditions associated with such genomic rearrangements, in particular cancers, such as lung cancer.

Abstract: A method for the analysis of minimal residual disease is provided. In some embodiments, the method comprises obtaining multiple pairs of primers designed to amplify sequences that contain a plurality of sequence variations that have been previously identified in a patient's tumor. Amplicons are then obtained through a targeted multiplex amplification that amplifies those sequences from cell-free DNA isolated from a plasma sample. The amplicons are sequenced and two or more of the sequence variations are detected from sequence reads, wherein the detecting comprises comparing a quantity of sequence reads containing a sequence variation against a threshold value. A score is then calculated for the patient sample based on the combined allele frequencies of the detected two or more sequence variations, wherein the score indicates the presence of minimal residual disease.

Abstract: A method of labelling a nucleic acid of interest (NAOI) is provided. In some embodiments, the method may comprise contacting a sample comprising the nucleic acid of interest with a pool of oligonucleotides, the pool comprising oligonucleotides having at least 5 different lengths; and attaching an oligonucleotide from the pool on to one or each end of the nucleic acid of interest, wherein attachment of an oligonucleotide moves the read start and/or stop coordinate when the labelled NAOI is sequenced.

Abstract: Provided herein is a method for sequence analysis that comprises analyzing PCR reactions that each contain different portions of the same sample, wherein at least some of the primer pairs are in more than one PCR reaction and at least one of the PCR reactions contains some but not all of the primer pairs of the other reaction(s).

Abstract: Provided herein, among other things, is a method of treating a cancer patient without the need for a tissue biopsy. In some embodiments, the method may comprise (a) performing or having performed a sequencing assay on cell-free DNA (cfDNA) from a sample of blood from the patient to determine if the cell-free DNA comprises actionable and/or non-actionable sequence variations in one or more target genes, and (b) treating the patient using the following method: i. administering a therapy that is targeted to an actionable sequence variation if the patient is identified as having the actionable sequence variation, and ii. administering a non-targeted therapy in the absence of any follow-up genetic testing on DNA extracted from a tissue biopsy if one or more non-actionable sequence variations and no actionable sequence variations are identified.

Abstract: The invention relates to methods for labelling individual nucleic acid molecules present in a sample, comprising contacting the nucleic acid molecules with an adaptor or mixture of adaptors, wherein the adaptor or adaptors comprise one or more universal nucleotide bases and a ligation moiety at their 3? end, and ligating an adaptor to the nucleic acid of interest, wherein the adaptor is ligated to the nucleic acid molecules at the 3? end of the adaptor. A random tag is then generated in situ by conducting an extension reaction over the ligated adaptor. Methods of the invention may be used to detect genetic alterations or variants in any nucleic acid with high specificity and high sensitivity, including mutations in nucleic acids such as ctDNA, cfDNA, and in viral, microbiome and plant nucleic acids. Methods of the invention may also be used in detection and correction of errors introduced into nucleic acids during processing.

Abstract: A method for analyzing cell free DNA (cfDNA) from the bloodstream of a cancer patient is provided. In some embodiments, the method may comprise sequencing at least part of the coding sequences of TP53 and KRAS in a sample of the cfDNA, analyzing the sequences to identify nucleotide transversions in the coding sequences of the genes, relative to reference sequences of the genes. In some embodiments, the method may comprise counting the total number of identified nucleotide transversions. The presence of nucleotide transversions indicates that the patient will be more responsive to the immune checkpoint inhibitor, whereas a decreased number of transversions or no transversios indicates that the patient will be less responsive to the immune checkpoint inhibitor.

Abstract: The present disclosure relates to methods for detecting and targeting genomic rearrangements, in particular gene fusion events, by targeting a DNA molecule of interest with a set or pool of primers, wherein the forward primers and reverse primers produce a PCR amplification product when a genomic rearrangement is present. The present disclosure also relates to methods of bioinformatic analysis to determine whether or not the detection of an amplification product from the selective PCR is actually indicative of the presence of a gene fusion. The present disclosure also related to related methods of diagnosis and treatment of diseases and conditions associated with such genomic rearrangements, in particular cancers, such as lung cancer.

Abstract: The present disclosure relates to methods for detecting and targeting genomic rearrangements, in particular gene fusion events, by targeting a DNA molecule of interest with a set or pool of primers, wherein the forward primers and reverse primers produce a PCR amplification product when a genomic rearrangement is present. The present disclosure also relates to methods of bioinformatic analysis to determine whether or not the detection of an amplification product from the selective PCR is actually indicative of the presence of a gene fusion. The present disclosure also related to related methods of diagnosis and treatment of diseases and conditions associated with such genomic rearrangements, in particular cancers, such as lung cancer.

Abstract: Provided herein is method for, among other things, estimating the number of sequence variations in a sample of DNA. In some embodiments, the method can be used to estimate the mutational load of a sample. In some embodiments, the method makes use of a set of primers that have 3? ends that specifically hybridizes to a sequence that is repeated multiple times in the genome. Thermocycling a reaction mix containing the primers may produce a reaction product comprising at least 50 amplicons having a total length of at least 100 kb. This product can be sequenced to provide an estimate of the number of sequence variations in the sample, and thus the mutational load of the sample.

Abstract: A method for analyzing cell free DNA (cfDNA) from the bloodstream of a cancer patient is provided. In some embodiments, the method may comprise sequencing at least part of the coding sequences of TP53 and KRAS in a sample of the cfDNA, analyzing the sequences to identify nucleotide transversions in the coding sequences of the genes, relative to reference sequences of the genes. In some embodiments, the method may comprise counting the total number of identified nucleotide transversions. The presence of nucleotide transversions indicates that the patient will be more responsive to the immune checkpoint inhibitor, whereas a decreased number of transversions or no transversions indicates that the patient will be less responsive to the immune checkpoint inhibitor.