Methods of labelling nucleic acids
AssigneeInivata Ltd.
InventorsSamuel WOODHOUSE, Tim FORSHEW, Stefanie Viola LENSING
Life of this patent
Estimating the term…
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.
Claims98
A method of labelling a nucleic acid of interest (NAOI), comprising:
- 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.
The method of any preceding claim, wherein each oligonucleotide in the pool comprises a spacer region, the pool comprising a mixture of oligonucleotides having at least 5 different lengths of spacer region.
The method of any preceding claim, wherein the oligonucleotides are attached to the NAOI by ligation, PCR, polymerase extension, isothermal/rolling circle amplification, loop-mediated isothermal amplification, or strand-displacement amplification.
The method of any preceding claim, wherein the oligonucleotides are attached to the NAOI by ligation.
The method of any preceding claim, wherein the pool of oligonucleotides comprises a mixture of oligonucleotides having at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or at least 50 different lengths of spacer region.
The method of any preceding claim, wherein the pool of oligonucleotides comprises a mixture of oligonucleotides having spacer regions of from 0 to 50 nucleotides, or from 0 to 25 nucleotides in length.
The method of any preceding claim, wherein the pool of oligonucleotides comprises a mixture of oligonucleotides having at least 10, different lengths of spacer region, wherein each spacer region is from about 0 to about 50 nucleotides in length.
The method of any preceding claim, wherein method comprises contacting a sample comprising a plurality of nucleic acids of interest with the pool of oligonucleotides, and further wherein oligonucleotides having spacer regions of at least 2 or at least 3 or at least 4 or at least 5 different lengths are attached to the nucleic acids of interest.
The method according to any preceding claim wherein spacer oligonucleotides of different lengths are present in the pool in an equimolar or substantially equimolar ratio.
The method of any preceding claim, wherein the longest spacer region in the pool of oligonucleotides is a reference sequence
The method of any preceding claim, wherein the spacer regions consist of a sequence according to X1Xn, wherein:
- a) X is any nucleotide;
- b) n is 0 to 75;
- c) the longest spacer region in the pool is a known reference sequence;
- d) each spacer region is a fragment of the reference sequence in the pool;
- and
- e) X1 is the first nucleotide in each spacer region.
The method of any preceding claim, where each oligonucleotide further comprises a universal priming site.
The method of any preceding claim, wherein the oligonucleotides are double-stranded and each strand of each oligonucleotide further comprises a universal priming site.
The method of any preceding claim, wherein the oligonucleotides in the pool are single stranded.
The method of any preceding claim, wherein the oligonucleotides in the pool are double stranded.
The method of any preceding claim, wherein the oligonucleotides comprise one or more universal bases.
The method of any preceding claim, wherein the oligonucleotide further comprises a filler region adjacent to the spacer region, wherein the filler region is a stretch of nucleotides between 1 and 20 nucleotides in length.
The method of any preceding claim, wherein the oligonucleotides further comprise an index sequence.
The method of any preceding claim, further comprising attaching an index sequence to the NAOI or the labelled NAOI.
The method of any preceding claim, wherein the NAOI is fragmented prior to attaching the oligonucleotide label.
The method of any preceding claim, wherein the NAOI is end-repaired using a mixture of enzymes prior to attaching the oligonucleotide label.
The method of any preceding claim, wherein the NAOI is amplified prior to attaching the oligonucleotide label.
The method of any preceding claim, wherein the nucleic acid of interest is contained in or derived from a patient sample, and the patient sample is processed prior to ligation of the nucleic acid of interest to an adaptor.
The method of 49, wherein processing of the sample comprises:
- a) purification of the sample to obtain a purified sample comprising the nucleic acid of interest;
- and/or
- b) extraction or isolation of the nucleic acid of interest from the patient sample;
The method of any preceding claim, wherein the method further comprises extracting the NAOI from the sample or enriching the sample for the NAOI prior to contacting the NAOI with the pool of oligonucleotide
The method of any preceding claim, further comprising amplifying the labelled NAOI, optionally wherein the amplification is performed using PCR.
The method of any preceding claim, wherein the method further comprises enriching the sample for the amplified NAOI or extracting the NAOI from the sample.
The method of any preceding claim, wherein the method comprises contacting the sample comprising the NAOI with multiple pools of oligonucleotides, wherein the longest spacer region in each pool is a reference sequence and further wherein the reference spacer region differs between pools.
The method of any preceding claim, wherein the method comprises contacting the sample comprising the NAOI with multiple pools of oligonucleotides, wherein each oligonucleotide comprises a filler region and further wherein the filler regions in a given pool of oligonucleotides are the same but the filler regions differ between pools.
The method of any preceding claim, wherein the method comprises performing replicate amplification reactions of the labelled NAOI.
The method of any preceding claim, further comprising sequencing the labelled and optionally amplified NAOI to provide a library of sequence reads.
The method of any preceding claim, further comprising determining the presence or absence of a genetic alteration in the nucleic acid of interest.
The method of any preceding claim, wherein the sample is a patient sample.
The method of any preceding claim, wherein the method comprises a step of obtaining the sample from a patient.
The method of any preceding claim, wherein the NAOI is contained in or derived from a patient sample, and the patient sample is processed prior to analysis.
The method according to any preceding claim wherein the length of the nucleic acid of interest is up to 50,000, up to 10,000, up to 5,000, up to 1,000, up to 500, up to 300, up to 250, up to 200, up to 180, up to 160, up to 140, up to 120, up to 100, up to 80, or up to 75 nucleotides in length.
The method according to any preceding claim wherein the length of the nucleic acid of interest is in the range of from:
- about 10 to about 10,000 nucleotides, about 10 to about 5,000 nucleotides, about 10 to about 3,000 nucleotides, about 10 to about 1,500 nucleotides about 10 to about 800 nucleotides about 10 to about 600 nucleotides about 10 to about 300 nucleotides, about 50 to about 250 nucleotides, about 100 to about 200 nucleotides, or about 100 to about 150 nucleotides.
The method according to any preceding claim, wherein the NAOI is single stranded.
The method according to any preceding claim, wherein the NAOI is double stranded.
The method according to any preceding claim, wherein the NAOI is viral nucleic acid, microbial nucleic acid or genomic nucleic acid.
The method according to any preceding claim, wherein the NAOI is DNA, RNA or cDNA.
The method according to any preceding claim, wherein the NAOI is DNA obtained by reverse transcriptase of RNA.
The method of any preceding claim, further comprising converting an RNA sequence to a DNA sequence to obtain the NAOI.
The method according to any preceding claim, wherein the target NAOI is a cell-free DNA (cfDNA)
The method of any preceding claim, wherein the nucleic acid of interest is at least 25 base pairs in length.
The method according to any preceding claim wherein the target NAOI is a ctDNA molecule of a cancer selected from the group consisting of acute lymphoblastic leukemia, acute or chronic lymphocyctic or granulocytic tumour, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoma, adrenal cancer, basal cell carcinoma, bone cancer, brain cancer, breast cancer, bronchi cancer, cervical dysplasia, chronic myelogenous leukemia, colon cancer, epidermoid carcinoma, Ewing's sarcoma, gallbladder cancer, gallstone tumour, giant cell tumour, glioblastoma multiforma, hairy-cell tumour, head cancer, hyperplasia, hyperplastic corneal nerve tumour, in situ carcinoma, intestinal ganglioneuroma, islet cell tumour, Kaposi's sarcoma, kidney cancer, larynx cancer, leiomyomater tumour, liver cancer, lung cancer, lymphomas, malignant carcinoid, malignant hypercalcemia, malignant melanomas, marfanoid habitus tumour, medullary carcinoma, metastatic skin carcinoma, mucosal neuromas, mycosis fungoide, myelodysplastic syndrome, myeloma, neck cancer, neural tissue cancer, neuroblastoma, osteogenic sarcoma, osteosarcoma, ovarian tumour, pancreas cancer, parathyroid cancer, pheochromocytoma, polycythemia vera, primary brain tumour, prostate cancer, rectum cancer, renal cell tumour, retinoblastoma, rhabdomyosarcoma, seminoma, skin cancer, small-cell lung tumour, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, thyroid cancer, topical skin lesion, veticulum cell sarcoma, and Wilm's tumour.
The method of any preceding claim, wherein the NAOI is a target gene.
Dependencies are inferred from the claim text.