# US 12,600,963 B2 · Depletion of abundant uninformative sequences

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- Number: US 12,600,963 B2
- Document ID: US12600963B2
- Status: Granted
- Kind: B2
- Application number: 17/153,019
- Filed: 2021-01-20
- Published: 2021-07-22
- Granted: 2026-04-14
- Earliest claimed: 2020-01-20
- Assignees: Tecan Genomics, Inc.
- Applicants: Tecan Genomics, Inc.
- Inventors: Bin Li; Lin Pham
- Web page: https://patentgrep.com/patent/US12600963B2

## CPC

- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6853: using modified primers or templates
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

The use of different primer sets in reverse transcription incorporates tags allowing for the selective amplification of cDNA transcribed using the different primers. Primers targeting non-desired RNA sequences such as ribosomal RNA can be used to prevent subsequent amplification of cDNA transcribed from those non-coding fragments. Accordingly effective depletion of non-desired sequences after cDNA amplification can be achieved. Systems and methods of the invention have applications in whole-transcriptome analysis. Non-coding sequence targeting primers can include nucleotide analogs that, when enzymatically processed, prevent subsequent amplification. Library preparation can include single primer isothermal amplification (SPIA) techniques wherein an RNA sequence required for SPIA is included in random primers but is absent from primers targeting non-coding RNA.

## Claims

### Claim 1

A method of preparing a cDNA library, the method comprising:
(i) providing RNA obtained from a sample, said RNA comprising a targeted subset of RNA sequences and a non-target subset of RNA sequences;
(ii) providing a first set of primers, wherein primers of the first set of primers are specific for the non-target subset of RNA sequences;
providing a second set of primers, wherein primers of the second set of primers are random primers or target-specific primers for the target subset of RNA wherein said second set of primers comprise a 5′ RNA tag,
(iii) transcribing the RNA into cDNA using the first and second set of primers;
(iv) selectively amplifying the cDNA transcribed from the targeted subset of RNA using the second set of primers thereby generating a cDNA library comprising amplicons from only said target subset of RNA, wherein the selectively amplifying step comprises amplifying the cDNA using an amplification primer targeting the 5′ tag or its compliment, wherein the amplification primer is a DNA/RNA chimeric primer,
wherein generating the cDNA library comprising amplicons from only said target subset of RNA does not include subtractive hybridization or enzymatic degradation of cDNA transcribed using the first set of primers.

### Claim 2 (depends on 1)

The method of claim 1 wherein the amplifying step comprises amplifying the cDNA using an archaeal family B DNA polymerase.

### Claim 3 (depends on 1)

The method of claim 1 wherein the cDNA library is a whole transcriptome cDNA library.

### Claim 4 (depends on 1)

The method of claim 1 wherein the first set of primers target ribosomal RNA (rRNA).

### Claim 5 (depends on 4)

The method of claim 4 wherein the first set of primers target 28S, 18S, 16S, or 12S rRNA.

### Claim 6 (depends on 1)

The method of claim 1 wherein the first set of primers target globin RNA.

### Claim 7 (depends on 1)

The method of claim 1 further comprising sequencing the cDNA library.

### Claim 8 (depends on 1)

The method of claim 1, wherein the second set of primers are target-specific primers for the target subset of RNA.

### Claim 9 (depends on 1)

The method of claim 1, wherein the 5′ tag comprises RNA and the cDNA amplification primer is a DNA/RNA chimeric single primer isothermal amplification (SPIA) primer.

### Claim 10 (depends on 9)

The method of claim 9, wherein the selective amplification is a single primer isothermal amplification.