# US 9,434,989 B2 · Methods and compositions for incorporating nucleotides

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- Number: US 9,434,989 B2
- Document ID: US9434989B2
- Status: Granted
- Kind: B2
- Application number: 14/691,042
- Filed: 2015-04-20
- Published: 2015-10-22
- Granted: 2016-09-06
- Earliest claimed: 2008-03-19
- Assignees: Intelligent Bio-Systems, Inc.
- Applicants: Intelligent BioSystems Inc.
- Inventors: Steven Gordon; Jerzy Olejnik
- Web page: https://patentgrep.com/patent/US9434989B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C07H 19/00: Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides ; Anhydro-derivatives thereof
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 21/05: Flow-through cuvettes (G01N21/09 takes precedence; handling fluid samples G01N1/10)
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/645: {Specially adapted constructive features of fluorimeters}
- G16B 40/10: Signal processing, e.g. from mass spectrometry [MS] or from PCR
- C12Q 2535/101: Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
- G01N 2021/0346: {Capillary cells; Microcells}
- G01N 2021/058: {Flat flow cell}
- G01N 2021/6417: {Spectrofluorimetric devices}
- G01N 2021/6419: {Excitation at two or more wavelengths}
- G01N 2021/6421: {Measuring at two or more wavelengths}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- G01N 2021/6441: {with two or more labels}
- G01N 2021/6471: {Special filters, filter wheel}
- G01N 2201/062: LED's
- G01N 2201/0627: Use of several LED's for spectral resolution
- G01N 2201/12: Circuits of general importance; Signal processing
- 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
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.

## Claims

### Claim 1

A method for determining an identity of a nucleotide at a position in a nucleotide sequence, comprising: a) providing primers, nucleic acid template molecules having a nucleotide sequence, polymerase, and a mixture of labeled and non-labeled reversible terminators, said labeled reversible terminators comprising a label linked to a nucleotide analogue, said label providing a signal, said signal having an intensity; b) incorporating, with a polymerase, a labeled reversible terminator from said mixture into a primer bound to a nucleic acid template molecule at a position corresponding to a complementary base in said template molecule; c) measuring the intensity of the signal of said label of said incorporated labeled reversible terminator; and d) determining the identity of the nucleotide at said position.

### Claim 2 (depends on 1)

The method of claim 1, wherein said mixture of labeled and non-labeled reversible terminators is reversibly terminated by an azidomethyl group at the 3′ position.

### Claim 3 (depends on 1)

The method of claim 1, wherein said labeled reversible terminators are labeled with at least one type of fluorescent dye and said measuring of step c) comprising measuring the intensity of said at least one type of fluorescent dye.

### Claim 4 (depends on 3)

The method of claim 3, wherein said intensity is measured in step c) in four channels.

### Claim 5 (depends on 4)

The method of claim 4, wherein said four channels are blue, green, yellow, and red channels.

### Claim 6 (depends on 3)

The method of claim 3, wherein said intensity is measured by a camera.

### Claim 7 (depends on 6)

The method of claim 6, wherein said intensity is solely dependent on the camera exposure and the concentration of said at least one type of fluorescent dye.

### Claim 8 (depends on 3)

The method of claim 3, wherein said intensity is measured in step c) in two channels.

### Claim 9 (depends on 8)

The method of claim 8, wherein said two channels are the green and yellow channels.

### Claim 10 (depends on 8)

The method of claim 8, wherein said two channels are the green and red channels.

### Claim 11

A method for determining an identity of a nucleotide at a position in a nucleotide sequence, comprising: a) providing a device capable of measuring color in more than one detector channels, primers, nucleic acid template molecules having a nucleotide sequence, polymerase, and a mixture of labeled and non-labeled reversible terminators, said labeled reversible terminators comprising a label linked to a nucleotide analogue, said label capable of being detected in two channels; b) incorporating, with a polymerase, a labeled reversible terminator from said mixture into a primer bound to a nucleic acid template molecule at a position corresponding to a complementary base in said template molecule; c) detecting the label of said incorporated labeled reversible terminator in two channels with said device; and d) determining the identity of the nucleotide at said position.

### Claim 12 (depends on 11)

The method of claim 11, wherein said mixture of labeled and non-labeled reversible terminators is reversibly terminated by an azidomethyl group at the 3′ position.

### Claim 13 (depends on 11)

The method of claim 11, wherein said labeled reversible terminators are labeled with at least one type of fluorescent dye and said detecting of step c) comprising detecting color of one type of fluorescent dye in two channels.

### Claim 14 (depends on 13)

The method of claim 13, wherein said at least one type of fluorescent dye is visible in the yellow channel and the green channel.

### Claim 15 (depends on 13)

The method of claim 13, wherein said at least one type of fluorescent dye is visible in the red channel and the green channel.

### Claim 16

A method for determining an identity of a nucleotide at a position in a nucleotide sequence, comprising: a) providing a device capable of measuring intensity in more than one detector channels, primers, nucleic acid template molecules having a nucleotide sequence, polymerase, and a mixture of labeled and non-labeled reversible terminators, said labeled reversible terminators comprising a label linked to a nucleotide analogue, said label providing a signal, said signal having an intensity; b) incorporating, with a polymerase, a labeled reversible terminator from said mixture into a primer bound to a nucleic acid template molecule at a position corresponding to a complementary base in said template molecule; c) detecting the intensity of the label of said incorporated labeled reversible terminator in two channels with said device; and d) determining the identity of the nucleotide at said position.

### Claim 17 (depends on 16)

The method of claim 16, wherein said mixture of labeled and non-labeled reversible terminators is reversibly terminated by an azidomethyl group at the 3′ position.

### Claim 18 (depends on 16)

The method of claim 16, wherein said labeled reversible terminators are labeled with at least one type of fluorescent dye and said detecting of step c) comprising detecting the intensity of one type of fluorescent dye in two channels.

### Claim 19 (depends on 16)

The method of claim 16, further comprising determining the ratio of the intensities in the two channels.