# US 11,111,520 B2 · Compositions and methods for enriching populations of nucleic acids

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- Number: US 11,111,520 B2
- Document ID: US11111520B2
- Status: Granted
- Kind: B2
- Application number: 15/157,374
- Filed: 2016-05-17
- Published: 2017-01-19
- Granted: 2021-09-07
- Earliest claimed: 2015-05-18
- Assignees: Karius, Inc.
- Applicants: Karius, Inc.
- Inventors: Timothy A. Blauwkamp; Fred Christians; Igor D. Vilfan; Scott Smith; Michael Kertesz
- Web page: https://patentgrep.com/patent/US11111520B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6888: for detection or identification of organisms
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

This disclosure provides methods and compositions that are useful for enriching a particular population of nucleic acids (a “population of interest”) within a complex mixture of nucleic acids. The population of interest may make up a minor portion of a complex mixture of nucleic acids. The methods and compositions provided herein are useful for detecting, predicting, diagnosing, or monitoring a disease or disorder, particularly a disease or disorder caused by a foreign microbe or pathogen.

## Claims

### Claim 1

A method of enriching microbial cell-free nucleic acids, the method comprising:
(a) identifying nucleic acid length ranges at which a ratio of microbial cell-free nucleic acid to host cell-free nucleic acid is favored;
(b) providing a host sample wherein the sample is selected from the group consisting of blood, plasma, serum, saliva, cerebrospinal fluid, synovial fluid, and lavage wherein the host sample comprises host cell-free nucleic acids and the microbial cell-free nucleic acids; and
(c) enriching the microbial cell-free nucleic acids relative to the host cell-free nucleic acids by enriching for nucleic acid length ranges at which a ratio of microbial cell-free nucleic acid to host cell-free nucleic acid is favored.

### Claim 2 (depends on 1)

The method of claim 1, comprising removing host cell-free DNA of one or more length intervals from the host sample.

### Claim 3 (depends on 1)

The method of claim 1, comprising isolating host cell-free DNA of one or more length intervals from the host sample.

### Claim 4 (depends on 2)

The method of claim 2, wherein the length intervals are selected from the group consisting of about 180 bases, about 360 bases, about 540 bases, about 720 bases, about 900 bases, and about 1000 bases.

### Claim 5 (depends on 2)

The method of claim 2, wherein the length ranges are length intervals selected from the group consisting of about 150 bases or multiples thereof, about 160 bases or multiples thereof, about 170 bases or multiples thereof, about 190 bases or multiples thereof, and any combination thereof.

### Claim 6 (depends on 1)

The method of claim 1, comprising isolating microbial cell-free nucleic acids that are up to about 150 bases in length from the host sample.

### Claim 7 (depends on 1)

The method of claim 1, further comprising performing a sequencing assay on the host cell-free nucleic acids and microbial cell-free nucleic acids from the host sample.

### Claim 8 (depends on 1)

The method of claim 1, wherein the host sample comprises at least five microbial cell-free nucleic acids and the method further comprises detecting the at least five microbial cell-free nucleic acids.

### Claim 9 (depends on 1)

The method of claim 1, wherein the host is human.

### Claim 10

A method of identifying microbes in a host, the method comprising:
(a) providing a sample selected from the group consisting of blood, plasma, serum, saliva, cerebrospinal fluid, synovial fluid, and lavage from the host;
(b) enriching the sample for microbial cell-free nucleic acids relative to host cell-free nucleic acids, wherein the enriching comprises enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges;
(c) analyzing the microbial cell-free nucleic acids; and
(d) identifying microbes in the host based on the microbial cell-free nucleic acids analyzed in (c).

### Claim 11 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially removing nucleic acids from the sample that are above about 150 bases in length.

### Claim 12 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially removing nucleic acids with lengths that are above about 300 bases in length from the sample.

### Claim 13 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially removing all nucleic acids from the sample that are above about 120 bases in length.

### Claim 14 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially enriching cell-free nucleic acids from the sample that are between about 10 bases and about 120 bases in length.

### Claim 15 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially digesting host derived nucleic acids.

### Claim 16 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially replicating the microbial cell-free nucleic acids.

### Claim 17 (depends on 10)

The method of claim 10, wherein the host is human.

### Claim 18 (depends on 1)

The method of claim 1, wherein the microbial cell-free nucleic acids are derived from pathogens.

### Claim 19 (depends on 18)

The method of claim 18, further comprising performing high throughput sequencing on the sample from the host.

### Claim 20 (depends on 18)

The method of claim 18, further comprising performing massively parallel sequencing on the host sample.

### Claim 21 (depends on 18)

The method of claim 18, wherein the microbial cell-free nucleic acids comprise DNA.

### Claim 22 (depends on 21)

The method of claim 21, wherein the microbial cell-free nucleic acids comprise bacterial, viral, fungal, or parasitic DNA, or a combination thereof.

### Claim 23 (depends on 18)

The method of claim 18, wherein the microbial cell-free nucleic acids comprise RNA.

### Claim 24 (depends on 23)

The method of claim 23, wherein the microbial cell-free nucleic acids comprise viral RNA.

### Claim 25 (depends on 1)

The method of claim 1, wherein the host cell-free nucleic acids or the microbial cell-free nucleic acids are not artificially fragmented.

### Claim 26 (depends on 25)

The method of claim 25, wherein the host cell-free nucleic acids or the microbial cell-free nucleic acids are not artificially enzymatically fragmented.

### Claim 27 (depends on 2)

The method of claim 2, wherein the one or more length intervals comprises between 110 and 1000 bases.

### Claim 28 (depends on 1)

The method of claim 1, wherein the cell-free nucleic acids comprise circulating cell-free nucleic acids.

### Claim 29 (depends on 1)

The method of claim 1, wherein the host sample is plasma.

### Claim 30 (depends on 10)

The method of claim 10, wherein the sample comprises circulating cell-free nucleic acids.

### Claim 31 (depends on 10)

The method of claim 10, wherein the sample is plasma.

### Claim 32 (depends on 1)

The method of claim 1, comprising removing host nucleic acids that are above about 150 bases in length from the host sample.

### Claim 33 (depends on 32)

The method of claim 32, comprising removing host cell-free DNA that is above about 150 bases in length from the host sample.

### Claim 34 (depends on 33)

The method of claim 33, wherein the host cell-free DNA that is above about 150 bases in length is above about 150 base pairs in length.

### Claim 35 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially enriching for nucleic acids with lengths that are below about 150 bases in length.

### Claim 36 (depends on 1)

The method of claim 1, comprising isolating microbial cell-free nucleic acids that are up to about 110 bases in length from the host sample.

### Claim 37 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially enriching cell-free nucleic acids from the sample that are between about 10 bases and about 110 bases in length.

### Claim 38 (depends on 1)

The method of claim 1, comprising isolating microbial cell-free nucleic acids that are up to about 70 bases in length from the host sample.

### Claim 39 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises preferentially enriching cell-free nucleic acids from the sample that are between about 10 bases and about 70 bases in length.

### Claim 40 (depends on 1)

The method of claim 1, comprising removing nucleosomal DNA length ranges or nucleosome-associated DNA length ranges.

### Claim 41 (depends on 1)

The method of claim 1, wherein the microbial cell-free nucleic acids are enriched at least 2-fold.

### Claim 42 (depends on 10)

The method of claim 10, wherein the microbial cell-free nucleic acids are enriched at least 2-fold.

### Claim 43 (depends on 1)

The method of claim 1, comprising removing nucleosomal DNA length ranges or nucleosome-associated DNA length ranges, wherein the microbial cell-free nucleic acids are enriched at least 2-fold.

### Claim 44 (depends on 40)

The method of claim 40, wherein removing nucleosomal DNA length ranges or nucleosomal-associated DNA length ranges comprises removing at least 50% of the first nucleosomal sized fragments.

### Claim 45 (depends on 40)

The method of claim 40, wherein the enriching comprises preferentially removing nucleic acids from the sample that are above about 150 bases in length.

### Claim 46 (depends on 10)

The method of claim 10, wherein enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises removing at least 50% of the first nucleosomal sized fragments.

### Claim 47 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises enriching for cell free nucleic acids based on DNA length ranges associated with nucleosomes.

### Claim 48 (depends on 10)

The method of claim 10, wherein the method further comprises extracting cell-free nucleic acids from the sample prior to the enriching in (b).

### Claim 49 (depends on 10)

The method of claim 10, wherein the enriching the sample for cell-free nucleic acids with length ranges that are not nucleosomal length ranges comprises using one or more antibodies specific to one or more histones.