UNIVERSAL METHOD FOR EXTRACTING NUCLEIC ACID MOLECULES FROM A DIVERSE POPULATION OF ONE OR MORE TYPES OF MICROBES IN A SAMPLE

• US 20180080065A1
• Filed: 11/28/2017
• Published: 03/22/2018
• Est. Priority Date: 09/15/2016
• Status: Active Grant

• Current Assignee: Sun Genomics, Inc.
• Original Assignee: Sun Genomics, Inc.
• Inventors: Jain, Suneer
• Granted Patent: US 10,428,370 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2035/115 Probiotics
• CPC Class Codes: A61K 35/741 Probiotics probiotic yeast, e.g. saccharomyces A61K36/06
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Abstract

Disclosed herein are methods of extracting genetic material from a diverse population of one or more types of microbes in a sample. Microbes can be prokaryotes or eukaryotes and may include bacteria, archaea, fungi, protozoa, helminths, parasites, viruses, phages, and others. Extraction may be from a single sample and subsequent identification may be through a molecular method such as qPCR, PCR, RFLP, SSCP, allele specific PCR, targeted sequencing, pull down sequencing, whole shotgun sequencing, or other methods.

Claims

1. A method for preparing a sample for analysis, which comprises:

a) mixing the sample with a first lysis solution comprising a detergent, and a chelator;

b) adding a second lysis solution having a lysozyme to the mixture of step a); and

c) adding a third lysis solution comprising a chaotropic agent, to the mixture of step b).

2. The method according to claim 1, wherein the first lysis solution further comprises one or more buffers, one or more mild detergents, and/or one or more proteases.

3. The method according to claim 1, wherein the third lysis solution further comprises a detergent such as SDS.

4. The method according to claim 3, wherein the third lysis solution comprises SDS at a working concentration of between about 0.1-10% w/v.

5. The method according to claim 1, wherein the chaotropic agent of the third lysis solution is lithium acetate and the mixture is then subjected to heat shock treatment.

6. The method according to claim 1, wherein after treatment with the third lysis solution, the mixture is treated with a fourth lysis solution comprising a second chaotropic agent, which may be the same or different from the chaotropic agent of the third lysis solution, and Proteinase K.

7. The method according to claim 6, wherein the second chaotropic agent is the same as the chaotropic agent of the third lysis solution.

8. The method according to claim 6, wherein the second chaotropic agent is different from the chaotropic agent of the third lysis solution.

9. The method according to claim 1, wherein the sample is subjected to a pretreatment step before treatment with the first lysis solution, said pretreatment step induces germination of any bacterial spores and/or fungal spores present in the sample.

10. The method according to claim 9, wherein the pretreatment step comprises mixing the sample with a mild detergent such as Tween-80.

11. The method according to claim 1, and further comprising a mechanical treatment step that causes physical lysis, said mechanical treatment step comprises sonication, bead mixing, bead mill homogenization, pressurization, microfluidization, and the like.

12. The method according to claim 1, and further comprising subjecting any genetic material extracted therefrom to metagenomics analysis.

13. The method according to claim 1, wherein the sample is of a commercially available probiotic or dietary supplement.

14. The method according to according to claim 1, wherein the sample is obtained from the gut of a subject.

15. A method of monitoring a probiotic treatment of a subject which comprises:

extracting genetic material from any microbes present in a first sample obtained from the subject, said genetic material extracted according to claim 1;

subjecting the genetic material extracted from the first sample to metagenomics analysis;

treating the subject with a probiotic and then extracting genetic material from any microbes present in a second sample obtained from the subject in the same manner as the extraction of genetic material from the first sample;

performing metagenomics analysis on the extracted genetic material from the second sample; and

comparing the results of the metagenomics analysis of the first sample with the metagenomics analysis of the second sample.

16. The method of claim 15, which further comprises modifying the probiotic treatment to obtain a desired microbe population within the subject.

17. The method of claim 15, further comprising analysis of metabolomic markers to determine appropriate probiotic treatment.

18. The method of claim 15, wherein probiotic treatment follows use of an antibiotic, chemotherapy, a pharmaceutical, environmental change, traveling, contaminate digestions, infarction of the intestines or gut, stress, or other effect which disruption of the microbial population can occur.

19. The method of claim 16, wherein modulation of the microbiome is to return an individual to a previous population of microbes of when the individual was known to be healthy.

20. The method of claim 16, wherein modulation of the microbiome by a probiotic and/or a prebiotic is to restore microbiome profile to a normal gut that has been defined either internally by a database or externally by a public database.

21. The method of claim 20, wherein normal is defined as similar to the microbiome profile of a fecal matter repository sample used in fecal matter transplants, but used as a probiotic/prebiotic to restore a dysbiosis.

22. The method of claim 15, wherein metagenomic analysis comprises use of a database having genomic data of organisms useful for identification of such organisms.

23. The method of claim 22, wherein the database may be processed as whole genomes, k-mers of various lengths that are common to a higher order and unique to a specific one, or other means of barcoding genomes to match them to sequencing results.

24. The method of claim 15, wherein metagenomic analysis comprises preprocessing of sequencing information selected from removing duplicates, removing adaptor sequencing, removing 5′

or 3′

sequencing to improve the quality of base calling, including only base calls of a particular quality (i.e., Q20 or greater), filtering human reads, creating paired reads or separating them, and limiting overlap of reads.

25. The method of claim 22, wherein metagenomic analysis comprises aligning sequencing information to the database by use of a software or system where the sequencing information may be broken into k-mers of particular length, used as full fragments, be scaffolded and aligned to a large reference genome, or other method to create a report of organisms identified, relative abundance of organism identified, genome size, total fragments aligned, unique fragments aligned at the strain, species, genus, family, order, class, phylum, kingdom, or domain.

26. The method of claim 15, wherein microbiome profiles enable identification of a disease, disorder, or specific signature indicating a dysbiosis where probiotic and/or dietary supplement treatment can be applied to modulate the microbiome to improve the underlying profile.

27. The method of claim 26, wherein a group is created based on demographic, phenotypic, or diagnostic information and a barcode assigned to that group of profiles.

28. The method of claim 27, wherein statistical analysis is used to determine how closely related the individual microbiome profile is to an known group in a database.

29. A computing system comprising:

a memory; and

one or more processors coupled to the memory, the one or more processors configured to perform operations to perform the method according to claim 15.

30. An automated platform for performing the method of claim 1.