Inivata Ltd.

Agency for Science, Technology and Research

Population Genetics Technologies Ltd.

Achilles Therapeutics UK Limited

Samuel Woodhouse

Tim Forshew

Stefanie Viola Lensing

Vincent Plagnol

Matthew Edward Smith

Karen Howarth

Michael Epstein

Andrew Lawson

Robert Osborne

Esther Musgrave-Brown

Giovanni Marsico

Duncan G Young

John Briant

Simon J Waddelow

Riccardo Rayan Siena

Toby Joseph Proctor

Joanne Wei-Gnan Tang

Matthew Parker

Michelle Griffin

Provided herein is a method for the analysis of minimal residual disease, the method comprising obtaining multiple pairs of primers, performing a targeted multiplex amplification using the multiple pairs of primers to produce amplicons, sequencing the amplicons or an amplification product thereof, thereby generating sequence reads, calculating a score based on the minority of sequences containing identified sequence variations, and using the calculated score to determine the presence or absence of minimal residual disease in the patient.

Method for the analysis of minimal residual disease

A method of sequencing a nucleic acid of interest (NAOI) is provided. In some embodiments, the method may comprise providing a sample from a patient, the sample having cell-free DNA molecules comprising NAOIs, amplifying the NAOIs, ligating adapters to the NAOIs, and sequencing the NAOIs. A method of labelling a NAOIs is also provided. In some embodiments, the method may comprise providing a sample from a patient having the NAOIs, amplifying the NAOIs, contacting the amplified NAOIs with a pool of oligonucleotides, attaching oligonucleotides to each of the amplified NAOIs to label the NAOIs. The method may further comprise sequencing the labelled NAOIs and grouping the resulting sequencing reads.

Method of sequencing a nucleic acid of interest

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.

Methods of labelling nucleic acids using variable length oligonucleotides

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.

Method for amplifying a genomic sample

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.

Methods of sequencing nucleic acids and error correction of sequence reads

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.

Computational method for detecting fusion events

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.

Medical device

Methods of labelling nucleic acids

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).

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&#x2032; 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.

Methods for labelling nucleic acids

Method for detecting a genomic rearrangement involving the ALK gene in cell-free DNA

Method for measuring mutational load

Providing herein, among other things, is a method for preparing a nucleic acid for sequencing. In some embodiments, the method comprises a) amplifying a nucleic acid template using a dNTP mix that contains 5-methyl dCTP, thereby producing product nucleic acid molecules that contains methylcytosines; b) digesting the product nucleic acid molecules with a methylation-dependent restriction endonuclease, thereby cleaving the product nucleic acid molecules at sites that are adjacent to at least some of the methylcytosine and producing fragments of the product nucleic acid molecules; and c) ligating double-stranded adaptors onto the ends of the fragments to produce adaptor-ligated products.

Growing community of inventors

Cambridge, United Kingdom

Samuel Woodhouse