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.
Claims20
A method for preparing target DNA duplexes for sequencing, said method comprising:
- (a) providing a plurality of sequencing adaptors each comprising a double-stranded polynucleotide duplex region containing a portion of a recognition sequence for a methyl-dependent endonuclease at its end, wherein a complete recognition sequence will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer;
- (b) covalently linking the duplex regions of said sequencing adaptors to first and second ends of a plurality of blunt-ended target DNA duplexes, thereby producing adaptor linked target DNA duplexes;
- and
- (c) removing adaptor dimers, if any, by digestion with a methyl-dependent endonuclease followed by digestion with one or more exonuclease(s).
An adaptor for polynucleotide sequencing, comprising a double-stranded polynucleotide duplex region containing a portion of a recognition sequence for a methyl-dependent endonuclease at its end, wherein a complete recognition sequence will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer.
An adaptor according to claim 25, wherein the first strand comprises a primer binding sequence on the single-stranded 3′ overhang region.
A method for sequencing DNA duplexes the method comprising:
- (a) providing a plurality of sequencing adaptors each comprising a double-stranded polynucleotide duplex region containing a portion of a recognition sequence for a methyl-dependent endonuclease at its end, wherein a complete recognition sequence will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer;
- (b) covalently linking the duplex regions of said sequencing adaptors to DNA duplexes, thereby producing adaptor linked DNA duplexes;
- (c) removing adaptor dimers, if any, by digestion with a methyl-dependent endonuclease followed by digestion with one or more exonuclease(s);
- and
- (d) sequencing the adaptor linked DNA duplexes.
Dependencies are inferred from the claim text.