CN-122003507-A - Amplification-free target enrichment workflow for direct detection of nucleic acid modifications

Abstract

The present disclosure provides methods of sequencing one or more target nucleic acid molecules, wherein each of the one or more target nucleic acid molecules comprises one or more modified nucleotides, and wherein the methods do not require conversion of any of the one or more modified nucleotides prior to sequencing, and/or do not require any amplification (PCR) cycles prior to sequencing. In some embodiments, sequencing is performed using a tag-side sequencing-by-sequencing device. In other embodiments, sequencing is performed using a single molecule real-time sequencing device.

Inventors

• C. Arnold
• NIELSEN CLINTON
• JIANG NAN
• SCHUBERT RALF
• BAYER DAVID CHRISTOPHER
• S. S. Chawan
• K. Di man
• H. Franklin
• S.He
• HOU YANLI
• S.J.Kang
• S. Krugez

Assignees

• 罗氏测序解决方案公司

Dates

Publication Date

20260508

Application Date

20241011

Priority Date

20231013

Claims (20)

1. 1. A method for nucleotide sequencing, the method comprising: a) Obtaining a sample comprising (i) one or more target nucleic acid molecules, wherein each of the one or more target nucleic acid molecules comprises one or more modified nucleotides, and (ii) one or more non-target nucleic acid molecules; b) Enriching the sample for the one or more target nucleic acid molecules, wherein no amplification cycle is performed before or after enrichment of the obtained sample; c) Obtaining a complex comprising a polymerase and a first target nucleic acid molecule from the one or more target nucleic acid molecules of the enriched sample; d) Contacting the obtained complex with a pool of labeled nucleotides to synthesize a first nascent polynucleotide, wherein the first nascent polynucleotide is complementary to at least a portion of the first target nucleic acid molecule, and wherein the presence of each labeled nucleotide in the first nascent polynucleotide generates one or more detectable events, and E) Correlating the generated one or more detectable events with a particular unmodified nucleotide or modified nucleotide, thereby determining the sequence of the first target nucleic acid sequence comprising the one or more modified nucleotides.
2. 2. The method of claim 1, wherein the one or more modified nucleotides in the one or more target nucleic acid molecules are not converted to a different modified nucleotide species prior to synthesis of the first novel polynucleotide.
3. 3. The method of claim 1, wherein one or more unmodified nucleotides in the one or more target nucleic acid molecules are not converted to uracil prior to synthesis of the first nascent polynucleotide.
4. 4. The method of claim 1, wherein the one or more modified nucleotides in the one or more target nucleic acid molecules are selected from the group consisting of 5-mC, 5-hmC, 4-mC, and 6-mA.
5. 5. The method of claim 1, wherein the one or more modified nucleotides in the one or more target nucleic acid molecules are selected from the group consisting of 5-fC, 4-mC, and 6-mA.
6. 6. The method of claim 1, wherein the one or more modified nucleotides in the one or more target nucleic acid molecules are selected from the group consisting of 5-caC, 4-mC, and 6-mA.
7. 7. The method of claim 1, wherein the one or more modified nucleotides in the one or more target nucleic acid molecules comprise 5-hmC nucleotides and/or 5-mC nucleotides, and wherein each of the 5-hmC and/or 5-mC nucleotides is converted to a 5-fC nucleotide and/or 5-caC nucleotide prior to synthesis of the first new polynucleotide.
8. 8. The method of any one of the preceding claims, wherein the obtained sample is enriched for the one or more target nucleic acid molecules by a hybrid capture process.
9. 9. The method of claim 8, wherein the hybridization capture process comprises (i) contacting the obtained sample with one or more probes to form target nucleic acid molecule-probe complexes, (ii) binding the formed target nucleic acid molecule-probe complexes to a solid support, (iii) removing non-target nucleic acid molecules from the obtained sample, and (iv) releasing the formed target nucleic acid molecule-probe complexes from the solid support.
10. 10. The method of any one of the preceding claims, further comprising generating a second strand for each of the one or more target nucleic acid molecules in the enriched sample.
11. 11. The method of claim 10, wherein the second strand is generated by primer extension.
12. 12. The method of any one of the preceding claims, further comprising ligating an adapter to each of the one or more target nucleic acid molecules in the enriched sample.
13. 13. The method of any one of the preceding claims, wherein the pool of labeled nucleotides comprises at least four different labeled nucleotides.
14. 14. The method of any one of the preceding claims, wherein the obtained composite further comprises nanopores within a membrane.
15. 15. The method of claim 14, wherein the obtained composite further comprises an electrode adjacent to the nanopore, wherein the electrode is adapted to measure the generated one or more detectable events.
16. 16. The method of claim 15, wherein the generated one or more detectable events comprise at least one of a residence time and a latency time.
17. 17. The method of claim 15, wherein the generated one or more detectable events comprise both a residence time and a latency time.
18. 18. The method of any one of claims 14 to 17, wherein the polymerase is associated with or coupled to the nanopore.
19. 19. A method according to any one of claims 1 to 3, wherein the polymerase is coupled to a solid support.
20. 20. The method of claim 19, wherein the solid support comprises a zero mode waveguide.

Description

Amplification-free target enrichment workflow for direct detection of nucleic acid modifications Technical Field The present disclosure relates to the field of nucleic acid-based diagnostics. More particularly, the invention relates to a method for detecting epigenetic modifications in a nucleic acid molecule, wherein the epigenetic modifications may have biological and clinical significance. Sequence listing This patent application contains a sequence listing that has been electronically filed in XML format and is hereby incorporated by reference in its entirety. The XML copy created at 2024, 9, 17 was named "P38402-US_sequence_Listing" and was 6,144 bytes in size. Background Nucleotide variants, particularly those that affect epigenetic function, provide basic regulatory information beyond simple genomic sequences. In eukaryotes, there are dynamic DNA epigenetic regulatory networks that include various modifications such as 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), 5-carboxycytosine (5-caC), 4-methylcytosine (4-mC) and 6-methyladenine (6-mA). For example, DNA methylation plays an important role in regulating various physiological and pathological processes in mammals. DNA methylation is an important epigenetic event that regulates embryo development, genomic imprinting, X inactivation, cell differentiation and proliferation. However, abnormal patterns of DNA methylation are associated with DNA instability and may lead to genetic disorders or cause acquired diseases such as cancer. Furthermore, DNA methylation is increasingly reported as a potential biomarker for other neurological and metabolic diseases. DNA methylation occurs predominantly at the C5 position within the cytosine loop within a cytosine-guanine (CpG) dinucleotide, often at gene regulatory sites such as promoter regions. Dense methylation of CpG in the promoter region of a gene is associated with dense chromatin structure, resulting in transcriptional silencing of the associated gene. If DNA hypermethylation occurs in the promoter region of some critical cancer-associated genes, it may lead to silencing of tumor suppressor genes and to tumorigenesis. DNA methylation changes are believed to exist and be detectable in tumors and blood. Thus, abnormal DNA methylation of a particular oncogene may be considered a biomarker for early diagnosis of cancer. Many methods have been developed to parse and analyze genomic DNA methylation. For example, bisulfite genomic sequencing provides a qualitative method for identifying 5-methylcytosine with single base pair resolution. The method is based on the discovery that deamination of cytosine and 5-methylcytosine (5-mC) results in different nucleobase transformations after sodium bisulfite treatment. The target nucleic acid is first treated with bisulphite reagents that specifically convert unmethylated cytosines to uracil residues while having no effect on methylated cytosines. The resulting uracil residues are then recognized as thymine in subsequent PCR amplification and sequencing, however, 5-mC is immune to this transformation and remains as cytosine allowing the 5-mC to be distinguished from unmethylated cytosine. The unwanted result of bisulfite conversion is that the reaction may be incomplete unless it is carried out at high temperatures for long periods of time, and this can degrade typically up to 95% of the DNA input. Furthermore, the bisulfite method cannot distinguish 5mC from closely related 5-hydroxymethylcytosine (5-hmC), another potential epigenetic biomarker. As unmethylated cytosine residues are converted to uracil residues and sequenced as thymine residues, the complexity of the sequence decreases, which may make mapping the sample on the genome more difficult. Methylation-sensitive restriction enzyme sequencing MSRE-Seq is a sequencing protocol that uses a methylation-sensitive restriction enzyme to analyze DNA methylation. These protocols require digestion of nucleic acid molecules with different methylation-sensitive restriction enzymes (e.g., bstUI, hpaII, notI and SmaI). Enzymatic digestion cleaves nucleic acid molecules at various unmethylated restriction sites, while methylation sites are protected from cleavage. Sequencing of the cleaved DNA fragments (enriched for unmethylated cpgs at their ends) allows recognition of cleavage sites. Although MSRE-Seq does not require bisulfite conversion, such methods have low resolution and low genome coverage. Fragmentation and tagging (tag) is the process of combining the fragmentation and adaptor incorporation steps. Ultra-high activity variants of bacterial Tn5 transposase that mediate fragmentation of double stranded DNA and ligate synthetic oligonucleotides have been widely employed in Next Generation Sequencing (NGS). DNA methylation sequencing methods based on tagging use transposases to fragment genomic DNA and attach sequencing adaptors in a single step. This method is more efficient than tra