Inivata Ltd.

Stefanie Viola Lensing

Samuel Woodhouse

Tim Forshew

Vincent Plagnol

Matthew Edward Smith

Karen Howarth

Michael Epstein

Giovanni Marsico

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

Methods of labelling nucleic acids

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.

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Cambridge, United Kingdom

Stefanie Viola Lensing