Methods and systems for high-depth sequencing of methylated nucleic acid

A methylated and unmethylated technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as high cost

Pending Publication Date: 2021-05-18
フリーノム ホールディングスインク
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This can be prohibitively costly in terms of plasma / cfDNA input and associated costs

Method used

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  • Methods and systems for high-depth sequencing of methylated nucleic acid
  • Methods and systems for high-depth sequencing of methylated nucleic acid
  • Methods and systems for high-depth sequencing of methylated nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0377] Example 1: Targeted EM sequencing library preparation and classifier generation

[0378] Starting material: 10-200 ng of double-stranded DNA.

[0379] 1. DNA preparation

[0380] EDTA was removed from the DNA before oxidation and the DNA sample had a final volume of 29 μl. Control DNA was used to assess oxidation and deamination. For sequencing on the Illumina platform, refer to the enzymatic methyl sequencing kit manual (NEB#E7120) for usage recommendations.

[0381] 2. Adapter ligation

[0382] 3. Oxidation of 5-methylcytosine and 5-hydroxymethylcytosine

[0383] TET2 buffer was prepared. TET2 Reagent was then added to a tube with TET2 Reaction Buffer Supplement followed by thorough mixing. On ice, add TET2 Reaction Buffer, Oxidation Supplement, Oxidation Promoter, and TET2 Enzyme directly to the DNA sample. The mixture was then mixed well by vortexing. After a brief centrifugation, the diluted Fe(II) solution was added to the mixture. The mixture was then mi...

Embodiment 2

[0395] Example 2: Targeted EM Sequencing Using a Transformation-Resistant Sequencing Adapter / Primer System

[0396] Identified adapters of known sequence are ligated to the ends of DNA molecules containing unknown sequences in the sample. The adapters are then used to PCR amplify the entire library of different molecules using a single primer set corresponding to the known adapters. The ligated adapter sequences also serve as binding sites for sequencing primers during subsequent sequencing reactions. To utilize the data provided by double-stranded sequencing, partially double-stranded adapters containing unique molecular identifiers (UMIs) were ligated to double-stranded DNA.

[0397] To improve the robustness, oxidation efficiency (and reduce cost) of EM-sequencing double-stranded sequencing, transformation-resistant adapters can be used to increase the consistency of sequencing library quality. Conversion-resistant adapters contain only unmodified bases and allow complete...

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Abstract

Methods and systems provided herein address current limitations of bisulfite-based methylation sequencing by improving the quality and accuracy of nucleic acid methylation sequencing and uses thereof for detection of disease. Methods that include minimally-destructive conversion methods for methylation sequencing as well as specialized UMI adapters provide for improved quality of sequencing libraries and sequencing information. Greater accuracy and more complete methylation-state information permits higher quality feature generation for use in machine learning models and classifier generation.

Description

[0001] cross reference [0002] This application claims the benefit of U.S. Provisional Application No. 62 / 855,795, filed May 31, 2019, which is hereby incorporated by reference in its entirety. [0003] Incorporate by reference [0004] All publications, patents and patent applications mentioned in this specification including the examples are herein incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference . In case of conflict, the present application, including any definitions herein, will control. Background technique [0005] Due to the stability of DNA and the role of DNA in normal differentiation and diseases such as cancer, DNA methylation can reflect tumor characteristics and phenotypic state, and thus has high potential for personalized medicine. Aberrant DNA methylation patterns occur early in the pathogenesis of cancer and thus may f...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6806C12Q1/6827C12Q1/6886C12Q2525/161C12Q2525/191C12Q2535/122C12Q2537/159C12Q2537/164C12Q2563/179C12Q2565/514C12Q1/6869C12Q1/6855C12Q1/686C12Q1/6876C12Q2600/154
Inventor 埃里克·阿里亚兹大卫·温伯格格雷格·霍根约翰·圣约翰迈克尔·皮尔森
Owner フリーノム ホールディングスインク
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