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KR-102961854-B1 - Analysis of multiple analytes using a single test

KR102961854B1KR 102961854 B1KR102961854 B1KR 102961854B1KR-102961854-B1

Abstract

The embodiments of the systems, methods, and compositions provided herein relate to a method for simultaneously analyzing multiple analytes within a single sample using a single assay. Some embodiments relate to the simultaneous analysis of DNA and RNA within a single sample, for example, the simultaneous creation of a DNA library and an RNA library.

Inventors

  • 스티머스 프랭크 제이.
  • 장 판
  • 포코로크 드미트리 케이.
  • 노어버그 스티븐

Assignees

  • 일루미나, 인코포레이티드

Dates

Publication Date
20260507
Application Date
20191125
Priority Date
20181130

Claims (20)

  1. A flow cell device for nucleic acid analysis comprising a polyT capture probe configured to hybridize with the 3' polyA tail of RNA and the 3' polyA tail tagged on DNA, and configured to simultaneously capture RNA and DNA, The above poly-T capture probe includes a first common sequence (CS1) sequence, and The above-mentioned poly-T capture probe is configured to confine the RNA and the DNA within a single compartment, enabling simultaneous analysis of RNA and DNA from a sample, and A flow cell device for nucleic acid analysis in which the above RNA is converted into DNA.
  2. A flow cell device for nucleic acid analysis according to claim 1, wherein the poly-T capture probe is immobilized in the flow cell device.
  3. A flow cell device for nucleic acid analysis according to claim 1, wherein the CS1 is configured to be used in downstream amplification, molecular indexing, or molecular demultiplexing.
  4. A flow cell device for nucleic acid analysis according to claim 1, wherein the sample is a cell population, a single cell, a nucleus population, or a single nucleus.
  5. A method for simultaneously preparing RNA and DNA libraries within a single compartment from a sample, A step of providing a sample comprising DNA and RNA, wherein the RNA comprises a 3' polyA tail; A step of tagmentating a 3' polyA tail to the DNA using a transposome having a polyA transposon; A step of contacting the sample with a polyT capture probe configured to hybridize to the 3' polyA tail of the RNA and the DNA within a single compartment, wherein the capture probe comprises a first common sequence (CS1); A step of hybridizing the capture probe to the RNA and the DNA to simultaneously capture DNA and RNA; A step of converting the above RNA into cDNA; and Step of simultaneously generating cDNA libraries and gDNA libraries A method including
  6. In paragraph 5, the method wherein the capture probe is fixed on a solid support.
  7. In claim 5, the method wherein the capture probe further comprises a substrate recognition sequence.
  8. In paragraph 5, the above sample is a cell population, a single cell, a cell nucleus population, or a single cell nucleus, method.
  9. In claim 6, the solid support is a substrate, an etched surface, a well, a covered well, a sealed well, an array, a flow cell device, a microfluidic channel, a bead, a magnetic bead, a column, a droplet, or a microparticle.
  10. A method according to claim 5, further comprising the step of simultaneously analyzing proteins in the single compartment, wherein the proteins are tagged.
  11. A method according to claim 5, wherein the sample comprises cells, and the cells are fixed with a fixative.
  12. A method according to claim 11, wherein the fixative comprises an alcohol or an aldehyde, wherein the alcohol comprises methanol and/or ethanol, and the aldehyde comprises formaldehyde, glutaraldehyde, and/or para-formaldehyde.
  13. As a kit for simultaneously analyzing DNA and RNA in a sample in a single compartment, Transposition reagents for tagging a 3' polyA tail to DNA; and A polyT capture probe configured to hybridize to the 3' polyA tail of the RNA and the DNA, comprising A kit in which the above capture probe includes a first common sequence (CS1), and the first CS1 identifies a common source of the DNA and RNA.
  14. In paragraph 13, the above-mentioned poly T capture probe is fixed on a solid support, in a kit.
  15. In claim 14, the solid support is a substrate, an etched surface, a well, a covered well, a sealed well, an array, a flow cell device, a microfluidic channel, a bead, a magnetic bead, a column, a droplet, or a microparticle, a kit.
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Description

Analysis of multiple analytes using a single test The systems, methods, and compositions provided herein relate to assays for simultaneously analyzing multiple analytes within a single sample. Specifically, the aspects disclosed herein relate to a method for analyzing DNA and RNA from a single sample in a single assay. The detection of specific nucleic acid sequences present in biological samples has been used as a method for, for example, the identification and classification of microorganisms, the diagnosis of infectious diseases, the detection and characterization of genetic abnormalities, the identification of genetic changes associated with cancer, the study of genetic susceptibility to diseases, and the measurement of responses to various types of treatments. A common technique for detecting specific nucleic acid sequences in biological samples is nucleic acid sequencing. Whole-genome sequencing, genotyping, targeted re-sequencing, gene expression, single-cell genomics, epigenomics, and protein expression analysis of tissue samples can be critical for identifying disease biomarkers, accurately diagnosing and prognosing diseases, and selecting appropriate treatments for patients. Often, this requires multiple assays to analyze specific analytes of interest, such as DNA, RNA, or proteins, individually. Various assays have been established to analyze these analytes separately and individually. However, the comprehensive analysis of multiple analytes is time-consuming and tedious. The present invention relates to a system, method, and composition for simultaneously analyzing multiple analytes in a sample using a single test. Some embodiments provided herein relate to nucleic acid libraries. In some embodiments, the library comprises a complementary DNA (cDNA) library and a genomic DNA (gDNA) library. In some embodiments, the cDNA library is derived from an mRNA molecule and comprises a nucleic acid having a first tag comprising a first barcode. In some embodiments, the gDNA library is derived from genomic DNA and comprises a nucleic acid having a second tag comprising a second barcode. In some embodiments, the first barcode and the second barcode are identical or different, and the first barcode and the second barcode identify a common source of the cDNA library and the gDNA library. In some embodiments, the cDNA library and the gDNA library are co-compartmented and prepared in the same environment. In some embodiments, the tag for DNA and the tag for RNA are identical. Some embodiments provided herein relate to a flowcell device. In some embodiments, the flowcell device comprises a first probe for capturing RNA and a second probe for capturing DNA, wherein the first probe comprises a first barcode and a first substrate recognition sequence and the second probe comprises a second barcode and a second substrate recognition sequence. In some embodiments, the first barcode and the second barcode are identical or different, and the first barcode and the second barcode identify a common source of RNA and DNA. In some embodiments, the first and second probes are configured to analyze RNA and DNA from a sample simultaneously in a single compartment. In the method of claim 19, the first and second capture probes are immobilized on a solid support. Some embodiments provided herein relate to a method for simultaneously analyzing DNA and RNA from a sample in a single compartment. In some embodiments, the method comprises the steps of providing a sample containing DNA and RNA—the RNA comprises a first tag—differentially tagging the DNA with a second tag, contacting the sample with a first capture probe for capturing RNA and a second capture probe for capturing the tagged DNA in a single compartment, hybridizing the first capture probe to the RNA and the second capture probe to the DNA to capture the RNA and DNA, and analyzing the DNA and RNA. In some embodiments, the first capture probe comprises a first barcode and the second capture probe comprises a second barcode, and the first barcode and the second barcode identify a common source of RNA and DNA. Some embodiments provided herein relate to a method for simultaneously generating nucleic acid libraries containing gDNA and cDNA in a single compartment. In some embodiments, the method comprises the steps of providing a sample containing DNA and RNA—the RNA comprises a first tag—differentially tagging the DNA with a second tag, contacting the sample with a first capture probe for capturing RNA and a second capture probe for capturing the tagged DNA in a single compartment, hybridizing the RNA and DNA to the first probe and the second probe, respectively, and simultaneously generating a cDNA library and a gDNA library from the hybridized RNA and DNA. In some embodiments, the first probe comprises a first barcode and the second probe comprises a second barcode, and the first barcode and the second barcode identify a common source of RNA and DNA. Some embodiments provid