Methods and compositions for preparing polynucleotides
AssigneeX Gen US CO.
InventorsYalei WU, Wai Ho LEE, Genhua ZHENG, Kai Qin LAO
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Abstract
Provided herein are methods, compositions, and kits for forming amplification products. In various embodiments provided herein, transposomes comprising transposases are used in forming tagged polynucleotides for downstream amplification and polynucleotide processing steps.
Claims23
A method of characterizing a polynucleotide in a sample, comprising:
- (a) contacting a double-stranded polynucleotide in a sample with a first set of transposome complexes, said first set comprising transposome complexes having a first transposon sequence, to generate a first plurality of overhang fragments that comprise the first transposon sequence at a 5′ end;
- (b) subjecting said first plurality of overhang fragments to an extension reaction to generate a first plurality of blunt-ended fragments;
- (c) conducting a primer-directed extension reaction with a primer comprising the first transposon sequence or a portion thereof to generate copies of the first plurality of blunt-ended fragments or fragments thereof;
- (d) contacting said first plurality of blunt-ended fragments or fragments thereof with a second set of transposome complexes, said second set comprising transposome complexes having a second transposon sequence, to generate a second plurality of overhang fragments that comprise the second transposon sequence at a 5′ end;
- (e) subjecting said second plurality of overhang fragments to an additional extension reaction to generate a second plurality of blunt-ended fragments;
- and
- (f) generating sequence reads from said second plurality of blunt-ended fragments or derivatives thereof, thereby characterizing the double-stranded polynucleotide in the sample.
A method of characterizing a polynucleotide in a sample, comprising:
- (a) contacting a double-stranded polynucleotide in a sample with a first set of transposome complexes, individual transposome complexes of said first set comprising a first transposon sequence, to generate a first plurality of overhang fragments that comprise the first transposon sequence at a 5′ end;
- (b) subjecting said first plurality of overhang fragments to an extension reaction to generate a first plurality of blunt-ended fragments;
- (c) conducting a primer-directed extension reaction with a primer comprising the first transposon sequence or a portion thereof to generate copies of the first plurality of blunt-ended fragments or fragments thereof;
- (d) contacting said first plurality of blunt-ended fragments or fragments thereof with a second set of transposome complexes, individual transposome complexes of said second set comprising a second transposon sequence, to generate a second plurality of overhang fragments that comprise the second transposon sequence at a 5′ end, wherein the first set of transposome complexes and the second set of transposome complexes are different in that:
- (i) transposon sequences comprised therein are distinct between the first and the second sets of transposome complexes;
- and/or
- (ii) one set of the transposome complexes utilized in step (a) or (d) are homodimer transposomes and one other set of the transposome complexes utilized are heterodimer transposomes;
- (e) subjecting said second plurality of overhang fragments to an additional extension reaction to generate a second plurality of blunt-ended fragments;
- and
- (f) generating sequence reads from said second plurality of blunt-ended fragments or derivatives thereof, thereby characterizing the double-stranded polynucleotide in the sample.
The method of any of the preceding claims, wherein said double-stranded polynucleotide comprises a chromosome.
The method of any of the preceding claims, wherein said double-stranded polynucleotide is from a single cell.
The method of any of the preceding claims, wherein characterizing said double-stranded polynucleotide yields haplotype information.
The method of any of the preceding claims, wherein characterizing said double-stranded polynucleotide comprises identifying a structural variation in said polynucleotide, wherein said structural variation is a copy number variation (CNV), an insertion, a deletion, a translocation, a retrotransposon, an inversion, a rearrangement, a repeat expansion, or a duplication.
The method of any of the preceding claims, wherein the double-stranded polynucleotide is at least about 1 megabase (Mb) in length.
The method of any of the preceding claims, wherein said first plurality of blunt-ended fragments comprises fragments of at least about 500 bases in length.
The method of any of the preceding claims, wherein said second plurality of blunt-ended fragments comprises fragments of at least about 100 bases in length.
The method of any of the preceding claims, wherein individual single-stranded polynucleotides of said first plurality of blunt-ended fragments form a stem-loop structure.
The method of any of the preceding claims, further comprising attaching sequencing adaptors to said second plurality of blunt-ended fragments to yield said derivatives thereof.
The method of any of the preceding claims, wherein individual transposome complexes of said first set and/or said second set comprise a transposase selected from Tn transposase, MuA transposase, and Vibhar transposase.
Dependencies are inferred from the claim text.