# US 11,441,169 B2 · Methods of small-RNA transcriptome sequencing and applications thereof

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- Number: US 11,441,169 B2
- Document ID: US11441169B2
- Status: Granted
- Kind: B2
- Application number: 16/308,912
- Filed: 2017-06-15
- Published: 2020-09-17
- Granted: 2022-09-13
- Earliest claimed: 2016-06-17
- Assignees: Ludwig Institute for Cancer Research
- Applicants: Ludwig Institute for Cancer Research Ltd
- Inventors: Omid R. Faridani; Rickard Sandberg
- Web page: https://patentgrep.com/patent/US11441169B2

## CPC

- C12Q 1/68: involving nucleic acids
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2525/191: incorporating an adaptor
- C12Q 2525/207: siRNA, miRNA
- C12Q 2535/122: Massive parallel sequencing
- C12Q 2537/159: Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
- C12Q 2537/163: blocking probe
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods and kits for sequencing and quantification of small RNA molecules from small quantities of starting materials, including single cells, are disclosed. The invention can be applied to, among others, preparing small RNA libraries, synthesizing cDNA from small RNAs, characterizing small RNAs from various cell types, studying cellular heterogeneity in pathological conditions, and diagnosing diseases.

## Claims

### Claim 1

A method of treating, detecting, or preparing an RNA sample comprising the steps of:
(a) providing a sample containing one or more ribosomal RNA molecules and one or more small RNA molecules;
(b) masking said ribosomal RNA molecules by providing one or more oligonucleotide(s) complementary to said ribosomal RNA molecules;
(c) contacting two or more 3′ oligonucleotide adaptors with a 3′ end of said small RNA molecules, wherein the 5′ end of the two or more 3′ oligonucleotide adaptors is adenylated and wherein the 3′ end of the two or more 3′ oligonucleotide adaptors is blocked by a first chemical group, to obtain a corresponding ligated 3′ oligonucleotide adaptor linked to said small RNA molecules and a corresponding unreacted free form 3′ oligonucleotide adaptor;
(d) hybridizing two or more RT primer oligonucleotides to said 3′ ligated oligonucleotide adaptor and said free form 3′ oligonucleotide adaptor, wherein the 5′ end of the RT primer is blocked by a second chemical group, and wherein said free form 3′ oligonucleotide adaptor is converted to a dimer of said free form 3′ oligonucleotide and said RT primer oligonucleotide;
(e) deadenylating the 5′ end of the two or more 3′ oligonucleotide adaptors;
(f) removing said dimer by digest with lambda exonuclease;
(g) ligating a 5′ oligonucleotide adaptor to a 5′ end of said small RNA molecules;
(h) performing reverse transcription of said small RNA molecules; and
(i) performing a first PCR reaction with a RT primer and a RP1 primer and a second PCR with RP1 and indexed primers.

### Claim 2 (depends on 1)

The method of claim 1, wherein said one or more oligonucleotides complementary to said small RNA molecules comprise a sequence selected from the group consisting of SEQ ID NOs: 1-11.

### Claim 3 (depends on 1)

The method of claim 1, wherein the one or more oligonucleotides complementary to said small RNA molecules comprise SEQ ID NO: 1.

### Claim 4 (depends on 1)

The method of claim 1, wherein one or both ends of said one or more oligonucleotides complementary to said small RNA molecules are blocked by a third chemical group.

### Claim 5 (depends on 4)

The method of claim 4, wherein the third chemical group is selected from the group consisting of a biotin moiety, an amino group, an inverted nucleotide, a dideoxy nucleotide, a spacer, a dye molecule moiety, a digoxigenin moiety, a cholesterol moiety, and a phosphate.

### Claim 6 (depends on 1)

The method of claim 1, wherein the 3′ oligonucleotide adaptor comprises SEQ ID NO: 82.

### Claim 7 (depends on 1)

The method of claim 1, wherein the RT primer oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 12-16.

### Claim 8 (depends on 1)

The method of claim 1, wherein the RT primer oligonucleotide comprises SEQ ID NO: 12.

### Claim 9 (depends on 1)

The method of claim 1, wherein the second chemical group is selected from the group consisting of a biotin moiety, an amino group, an inverted nucleotide, a dideoxy nucleotide, a spacer, a dye molecule moiety, a digoxigenin moiety, a cholesterol moiety, and a phosphate.

### Claim 10 (depends on 1)

The method of claim 1, wherein the 5′ adaptor comprises a unique molecular identifier.

### Claim 11 (depends on 1)

The method of claim 1, wherein the 5′ oligonucleotide adaptor comprises SEQ ID NOs: 17-33.

### Claim 12 (depends on 1)

The method of claim 1, wherein the 5′ oligonucleotide adaptor comprises SEQ ID NO: 17.

### Claim 13 (depends on 1)

The method of claim 1, wherein said RP1 primer comprises SEQ ID NO: 83.

### Claim 14 (depends on 1)

The method of claim 1, wherein said indexed primers comprise a sequence selected from the group consisting of SEQ ID NOs: 34-81.

### Claim 15 (depends on 1)

The method of claim 1, wherein the first chemical group is dideoxycytidine (ddC).

### Claim 16 (depends on 9)

The method of claim 9, wherein the second chemical group is biotin.