Highly multiplexed phylogenetic imaging of microbial communities

Patent metadata

Field	Value
Publication number	US-12410425-B2
Application number	US-201916978891-A
Country	US
Kind code	B2
Filing date	Mar 7, 2019
Priority date	Mar 8, 2018
Publication date	Sep 9, 2025
Grant date	Sep 9, 2025

Abstract

Micron scale biogeography is a major driver of physiology and ecology of complex microbial biofilm communities, which remains elusive largely due to the lack of tools for spatially resolved phylogenetic mapping. This disclosure provides methods, computer-readable storage devices and kits that allow highly multiplexed and spatially resolved imaging of microbial community spatial organization. The disclosure provides a highly-multiplexed approach to resolve the spatial structure of complex microbial community at high taxonomic resolution.

First claim

What is claimed is:

1. A method comprising:
providing a list of taxa of microorganisms, wherein each taxon in the list is assigned a unique n-bit binary code selected from a plurality of unique n-bit binary codes, and wherein n is an integer equal or greater than 10;
providing a set of n number of decoding probes, wherein each decoding probe
corresponds to a digit in the plurality of unique n-bit binary codes,
is conjugated with a label that provides a detectable signal, wherein the labels on the decoding probes are different from each other, and
is substantially complementary to a readout sequence selected from a set of n number of readout sequences;

providing a set of encoding probes, wherein the set of encoding probes includes a plurality of subsets of encoding probes, wherein
each encoding probe comprises a targeting sequence and one or more readout sequences,
the encoding probes within each subset comprise a targeting sequence that is specific to a taxon in the list of taxa of microorganisms and is different from a targeting sequence of the encoding probes of another subset, and
the readout sequences in the encoding probes within a subset are selected from the set of n number of readout sequences based on the unique n-bit binary code assigned to the taxon which the targeting sequence of the subset is specific to;

providing a sample suspected of comprising one or more of the taxa of microorganisms;
contacting the sample with the set of encoding probes to permit hybridization of the targeting sequences to the corresponding taxa of microorganisms present in the sample;
subsequently contacting the sample with the set of decoding probes to permit hybridization of one or more decoding probes to the readout sequences in encoding probes that are bound to the sample;
detecting the decoding probes that are bound to the sample after a single round of two-step hybridization with the encoding probes and decoding probes;
determining the unique binary codes based on the detected decoding probes; and
identifying the taxa of microorganisms present in the sample based on the determined unique binary codes.

2. The method of claim 1 , wherein the targeting sequence for a subset of encoding probes is substantially complementary to a consensus 16S ribosomal sequence specific to a taxon.

3. The method of claim 1 , wherein the encoding probes within each subset comprise a plurality of targeting sequences, wherein the plurality of targeting sequences are specific to the same taxon.

4. The method of claim 1 , wherein the list of taxa of microorganisms is selected from a list of phyla, a list of classes, a list of orders, a list of families, a list of genera, or a list of species.

5. The method of claim 1 , wherein the label is a fluorophore.

6. The method of claim 5 , wherein the fluorophore is selected from the group consisting of Alexa 405, Pacific Blue, Pacific Green, Alexa 488, Alexa 532, Alexa 546, Rhodamine Red X, Alexa 610, Alexa 647, DyLight-510-LS, Hydroxycoumarin, methoxycoumarin, Cy2, FAM, Flourescein FITC, Alexa 430, R-phycoerythrin (PE), Tamara, Cy3.5 581, Rox, Alexa fluor 568, Red 613, Texas Red, Alexa fluor 594, Alexa fluor 633, Alexa fluor 660, Alexa fluor 680, Cy5, Cy 5.5, Cy 7, and Allophycocyanin.

7. The method of claim 1 , wherein the detecting is achieved by a microscope.

8. The method of claim 1 , wherein the determining is achieved by a support vector machine.

9. The method of claim 1 , wherein the sample is selected from an environmental sample or a biological sample.

10. The method of claim 9 , wherein the biological sample is selected from the group consisting of bronchoalveolar lavage fluid (BAL), blood, serum, plasma, urine, cerebrospinal fluid, pleural fluid, synovial fluid, peritoneal fluid, amniotic fluid, gastric fluid, lymph fluid, interstitial fluid, tissue homogenate, a tumor, cell extracts, saliva, sputum, stool, physiological secretions, tears, mucus, sweat, milk, semen, seminal fluid, and vaginal secretions.

11. The method of claim 1 , wherein the sample is a solid sample or a liquid sample.

12. The method of claim 1 , wherein the sample is washed after each hybridization with encoding probes and decoding probes.

13. The method of claim 1 , wherein each decoding probe comprises at least 10 nucleotides.

14. The method of claim 1 , wherein each targeting sequence comprises at least 15 nucleotides.

Assignees

• Univ Cornell

Inventors

• Shi Hao
• De Vlaminck Iwijn

Classifications

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C40B20/00

  Methods specially adapted for identifying library members · CPC title

• C12N1/00

  Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor · CPC title

• G16B25/00

  ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression · CPC title

• C12N15/1089

  Design, preparation, screening or analysis of libraries using computer algorithms · CPC title