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US 11,519,026 B2Granted
Methods for removal of adaptor dimers from nucleic acid sequencing preparations
AssigneeRoche Sequencing Solutions, Inc.
InventorsArkadiusz Bibillo, Randall W. Davis
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Abstract
Sequencing adaptors and methods are provided for preparation of polynucleotides for sequencing. The sequencing adaptors contain a portion of a recognition sequence for a methyl-dependent endonuclease. Unwanted adaptor dimers that form during ligation of adaptors to target polynucleotides produce a complete restriction sequence and are cleaved by the endonuclease, followed by exonuclease digestion, thereby removing the dimers.
Claims18
A method for preparing target DNA duplexes for sequencing, wherein the method comprises:
- (a) a step of either:
- (i) extending the target DNA duplexes with standard unmethylated nucleotide bases, or (ii) amplifying the target DNA duplexes with standard unmethylated nucleotide bases;
- (b) providing a plurality of sequencing adaptors, wherein each of the plurality of sequencing adaptors comprises a single-stranded polynucleotide hairpin region and a double-stranded polynucleotide duplex region, wherein the double-stranded polynucleotide duplex region contains a portion of a recognition sequence for a methyl-dependent endonuclease at one of its ends, and wherein a complete recognition sequence will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer;
- (c) covalently linking the duplex regions of the sequencing adaptors to first and second ends of a plurality of blunt-ended target DNA duplexes, thereby producing adaptor-linked target DNA duplexes;
- and
- (d) removing adaptor dimers, if any, by digestion with a methyl-dependent endonuclease, followed by digestion with one or more exonuclease(s).
3 more elements
A method for sequencing DNA duplexes, wherein the method comprises:
- (a) a step of either:
- (i) extending the target DNA duplexes with standard unmethylated nucleotide bases, or (ii) amplifying the target DNA duplexes with standard unmethylated nucleotide bases;
- (b) providing a plurality of sequencing adaptors, wherein each of the plurality of sequencing adaptors comprises a double-stranded polynucleotide duplex region, wherein the double-stranded polynucleotide duplex region contains a portion of a recognition sequence for a methyl-dependent endonuclease at its end, and wherein a complete recognition sequence will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer;
- (c) covalently linking the double-stranded polynucleotide duplex regions of the plurality of sequencing adaptors to target DNA duplexes, thereby producing adaptor-linked DNA duplexes;
- (d) removing adaptor dimers, if any, by digestion with a methyl-dependent endonuclease followed by digestion with one or more exonuclease(s);
- and
- (e) sequencing the adaptor linked DNA duplexes, and wherein either:
- (1) each of the plurality of sequencing adaptors further comprises a single-stranded polynucleotide hairpin region, or
- (2) each of the plurality of sequencing adaptors is a linear polynucleotide comprising a first and second nucleotide strand, wherein each of the plurality of sequencing adaptors comprises:
- (i) a first end comprising the double-stranded polynucleotide duplex region, and (ii) a second end comprising a 3′ end of the first polynucleotide strand and a 5′ end of the second polynucleotide strand, wherein the first polynucleotide strand comprises a single-stranded 3′ overhang region comprising a thionated nucleotide at its 3′ end, and wherein the second polynucleotide strand comprises a thionated nucleotide at its 5′ end.
7 more elements
A method for preparing target DNA duplexes for sequencing, wherein the method comprises:
- (a) a step of either:
- (i) extending the target DNA duplexes with standard unmethylated nucleotide bases, or (ii) amplifying the target DNA duplexes with standard unmethylated nucleotide bases;
- (b) providing a plurality of sequencing adaptors, wherein each of the plurality of sequencing adaptors comprises a double-stranded polynucleotide duplex region, wherein the double-stranded polynucleotide duplex region contains a portion of a recognition sequence for a methyl-dependent endonuclease at one of its ends, and wherein a complete recognition sequence will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer;
- (c) covalently linking the duplex regions of the sequencing adaptors to first and second ends of a plurality of blunt-ended target DNA duplexes, thereby producing adaptor-linked target DNA duplexes;
- and
- (d) removing adaptor dimers, if any, by digestion with a methyl-dependent endonuclease, followed by digestion with one or more exonuclease(s), and wherein
- each of the plurality of sequencing adaptors is a linear polynucleotide comprising a first and second polynucleotide strand, wherein each of the plurality of sequencing adaptors comprises:
- (i) a first and comprising the double-stranded polynucleotide duplex region, and (ii) a second end comprising a 3′ end of the first polynucleotide strand and a 5′ end of the second polynucleotide strand, wherein the first polynucleotide strand comprises a single-stranded 3′ overhang region comprising a thionated nucleotide at its 3′ end, and wherein the second polynucleotide comprises a thionated nucleotide at its 5′ end.
5 more elements
Dependencies are inferred from the claim text.