FIELD OF THE INVENTION

The invention relates to methods and compositions for removal of unwanted adaptor dimers from a nucleic acid sequencing sample preparation mixture.

BACKGROUND

Major DNA sequencing technologies rely on the attachment of specific DNA adaptors to the ends of sample DNA fragments to create polymerase primer sites, to add sample capture sites, to “barcode” samples, and to add calibration sites to samples, among many possible uses. Adaptor nucleic acids are ligated to each end of a DNA sample to be sequenced. The creation of adaptor ligated DNA fragments often include adaptors ligating to each other, forming unwanted “adaptor dimers.”

Currently, adaptor dimers are removed by small fragment cleanup steps, such as use of magnetic beads to capture and separate large polynucleotide fragments from small fragments. Such a method is not sequence based and is therefore nonspecific and inefficient. If the sample is circularized, non-circularized adaptors may be depolymerized from their exposed ends by treatment with exonucleases. In particular, the removal of circularized adaptor dimers from a set of near- or similar-sized DNA sample fragments is especially difficult. Cleanup steps do not efficiently remove the dimers based on size or take considerable time. Further, circular adaptors cannot be depolymerized by exonucleases since they do not have an exposed end.

More efficient and specific methods for removal of unwanted adaptor dimers are needed.

BRIEF SUMMARY OF THE INVENTION

Adaptors for polynucleotide sequencing are provided as well as methods and kits for their use in sequencing methods. The adaptors and methods described herein may be used to produce polynucleotide samples for sequencing without adaptor dimers or with very low levels of adaptor dimers, which would otherwise interfere with or reduce efficiency of polynucleotide sequencing methods.

In one aspect, a method is provided for preparing target DNA duplexes for sequencing. In the methods disclosed herein, adaptors include a portion of a methyl-dependent endonuclease recognition sequence, which when covalently joined in an adaptor dimer, will form the complete recognition sequence and will be subject to digestion by the endonuclease. Adaptor dimers are removed by digestion by the methyl-dependent endonuclease, followed by exonuclease digestion. Adaptors linked to target polynucleotides for sequencing are not sensitive to digestion by the endonuclease and exonuclease enzymes, and thus only the unwanted adaptor dimers are removed from the sample.

In one embodiment, the method includes: (a) covalently linking double-stranded polynucleotide duplex regions of a plurality sequencing adaptors to first and second ends of a plurality of blunt-ended target DNA duplexes, thereby producing a plurality of adaptor-linked target DNA duplexes with a sequencing adaptor covalently linked at each end of the target DNA duplex, wherein the double-stranded polynucleotide duplex region of each adaptor includes a portion of a recognition sequence for a methyl-dependent endonuclease at its end, wherein a complete recognition sequence for the methyl-dependent endonuclease will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer; and (b) removing adaptor dimers, if any, by digestion with a methyl-dependent endonuclease followed by digestion with one or more exonuclease(s).

In another embodiment, the method includes: (a) providing a plurality of sequencing adaptors, wherein each of said adaptors includes a double-stranded polynucleotide duplex region, wherein the double-stranded polynucleotide duplex region includes a portion of a recognition sequence for a methyl-dependent endonuclease at its end, wherein a complete recognition sequence for the methyl-dependent endonuclease 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 a plurality of adaptor-linked target DNA duplexes with a sequencing adaptor covalently linked at each end of the target DNA duplex; and (c) removing adaptor dimers, if any, by digestion with a methyl-dependent endonuclease followed by digestion with one or more exonuclease(s).

The adaptors may include a single-stranded hairpin region or may be linear. In one embodiment, each of the adaptors includes a single-stranded polynucleotide hairpin region and the double-stranded polynucleotide duplex region. In another embodiment, each of the adaptors is a linear polynucleotide that includes first and second polynucleotide strands, wherein each adaptor includes the double-stranded polynucleotide duplex region and a 3′ overhang region, wherein the first strand includes the 3′ overhang region and includes a modified nucleotide that is resistant to exonuclease digestion (e.g., a thionated nucleotide) at or near the 3′ end and the second strand, which is part of the polynucleotide duplex, includes a modified nucleotide that is resistant to exonuclease digestion (e.g., a thionated nucleotide) at or near the 5′ end.

In some embodiments, the double-stranded polynucleotide duplex region in each of the adaptors includes a first strand with a 5′ end hybridized to a second strand with a 3′ end, wherein the first strand includes the sequence GG at the 5′ end hybridized to the sequence CMeC at the 3′ end of the second strand. The methyl-dependent endonuclease may be, for example, MspI or MspII, wherein adaptor dimers include the recognition sequence CCMeGG.

In some embodiments, the double-stranded polynucleotide duplex region in each of the adaptors includes a first strand with a 5′ end hybridized to a second strand with a 3′ end, wherein the first strand includes the sequence TC at the 5′ end hybridized to the sequence GAMeat the 3′ end. The methyl-dependent endonuclease may be, for example, DpnI or DpnII, wherein adaptor dimers include the recognition sequence GAMeTC.

In some embodiments, the exonuclease(s) used for digestion of cleaved adaptor dimers includes Exonuclease VII, Exonuclease III, and/or T5 Exonuclease. In some embodiments, Exonuclease III and Exonuclease VII are used. In some embodiments, Exonuclease III and T5 Exonuclease are used.

In some embodiments, a ligase enzyme is used to covalently link the double-stranded polynucleotide duplex regions of the sequencing adaptors to the first and second ends of the blunt-ended target DNA duplexes.

In various embodiments, the target DNA duplexes may include amplified target polynucleotides, non-amplified fragments of genomic DNA, copies of fragments of genomic DNA synthesized with unmethylated nucleotides, and/or cDNA transcribed from fragments of RNA. In various embodiments, the target DNA duplexes may be derived from a biological fluid or tissue sample and/or from one or more microorganism(s).

In another aspect, a method is provided for preparing a polynucleotide sample for sequencing, which method includes covalently attaching sequencing adaptors to target DNA duplexes to be sequenced, in accordance with the methods described herein, and wherein the resulting polynucleotide sample for sequencing includes less than about 1% adaptor dimers.

In another aspect, a method is provided for preparing a polynucleotide sample for sequencing, which method includes covalently attaching sequencing adaptors to target DNA duplexes to be sequenced, in accordance with the methods described herein, including hybridizing a primer to a primer binding sequence on the covalently linked adaptors and extending the primer with a DNA polymerase enzyme, thereby preparing a primer extension product for sequencing. For example, the primer may be extended to produce a complementary copy of one strand of the target DNA duplex, wherein the copy is sequenced as it is synthesized by the polymerase. In one embodiment, the primer binding sequence is in a single-stranded hairpin region of the adaptor. In another embodiment, the primer binding sequence is in a 3′ single-stranded overhang region of the adaptor.

In another aspect, adaptors are provided for polynucleotide sequencing. The adaptor includes a double-stranded polynucleotide duplex region, wherein the double-stranded polynucleotide duplex region includes a portion of a recognition sequence for a methyl-dependent endonuclease at its end, and wherein a complete recognition sequence for the methyl-dependent endonuclease will be formed if the duplex regions of two adaptors are covalently linked together to produce an adaptor dimer.

In an embodiment, the adaptor includes a single-stranded polynucleotide hairpin region and the double-stranded polynucleotide duplex region, optionally including a primer binding site in the hairpin region.

In an embodiment, the adaptor is a linear polynucleotide that includes first and second polynucleotide strands, wherein the adaptor includes the double-stranded polynucleotide duplex region and a 3′ overhang region, wherein the first strand includes the 3′ overhang region and includes a modified (e.g., thionated) nucleotide that is resistant to exonuclease digestion at or near the 3′ end and the second strand, which is part of the polynucleotide duplex, includes a modified (e.g., thionated) nucleotide that is resistant to exonuclease digestion at or near the 5′ end.

In an embodiment, the double-stranded polynucleotide duplex in the adaptor includes a first strand with a 5′ end hybridized to a second strand with a 3′ end, wherein the first strand includes the sequence GG at the 5′ end hybridized to the sequence CMeC at the 3′ end of the second strand.

In an embodiment, the double-stranded polynucleotide duplex in the adaptor includes a first strand with a 5′ end hybridized to a second strand with a 3′ end, wherein the first strand includes the sequence TC at the 5′ end hybridized to the sequence GAMeat the 3′ end.

In another aspect, a method is provided for sequencing polynucleotides, the method including sequencing a polynucleotide sample that includes a plurality of adaptor-linked DNA duplexes with a sequencing adaptor as described herein covalently linked at each end of the target DNA duplex, wherein the polynucleotide sample includes less than about 1% adaptor dimers.

In another aspect, a polynucleotide sample for sequencing is provided, the sample including a plurality of adaptor-linked DNA duplexes with a sequencing adaptor as described herein covalently linked at each end of the target DNA duplex, wherein the polynucleotide sample includes less than about 1% adaptor dimers.

In another aspect, a kit is provided for polynucleotide sequencing. In some embodiments, the kit includes: (a) a plurality of sequencing adaptors as described herein; and (b) instructions for preparing target DNA duplexes for sequencing as described herein. In some embodiments, the kit further includes (c) a ligase enzyme; (d) a methyl dependent endonuclease enzyme; (e) one or more exonuclease enzyme(s); and/or (f) one or more sequencing primer(s).

