Method of improved sequencing by strand identification
AssigneeAccuragen Holdings Limited
InventorsMalek Faham, Shengrong Lin, Ling Fung Tang, Yontao Lu, Zhaohui Sun, Yingyu Wang, Li Weng
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Abstract
In some aspects, the present disclosure provides methods for identifying sequence variants, as well as methods of determining copy number of a genetic locus in a sample. Systems and kits for performing methods of the disclosure, as well as compositions produced by or useful in methods of the disclosure are also provided. In some embodiments, methods comprise extending 3′ ends of polynucleotides by adding one or more pre-determined nucleotides. In some embodiments, methods comprise use of a strand-tagging sequence.
Claims16
A method of identifying complementary strands in a nucleic acid sample comprising a plurality of double-stranded polynucleotides, each double-stranded polynucleotide of the plurality comprising a first complementary strand and a second complementary strand, each having a 5′ end and a 3′ end, the method comprising:
- (a) modifying a polynucleotide sequence of at least one of a first complementary strand and a second complementary strand of individual double-stranded polynucleotides by adding one or more predetermined nucleotides in a base by base manner to the 3′ end of at least one of the first complementary strand and the second complementary strand;
- (b) circularizing a plurality of first complementary strands and a plurality of second complementary strands to form a plurality of single stranded polynucleotide circles, the one or more predetermined nucleotides defining a boundary between 3′ sequences and 5′ sequences of each single stranded polynucleotide circle;
- (c) amplifying by rolling circle amplification the single stranded polynucleotide circles to form a plurality of concatemers;
- (d) sequencing said plurality of concatemers, to yield a plurality of sequencing reads;
- and
- (e) identifying from the plurality of sequencing reads, a given first complementary strand and a given second complementary strand as originating from a common double-stranded polynucleotide based on (i) sequences of the respective 3′ ends and 5′ ends adjacent to the one or more predetermined nucleotides, and (ii) sequences of the one or more predetermined nucleotides.
A method of identifying a sequence variant in a nucleic acid sample comprising a plurality of double-stranded polynucleotides, each double-stranded polynucleotide of the plurality comprising a first complementary strand and a second complementary strand, each having a 5′ end and a 3′ end, the method comprising:
- (a) modifying a polynucleotide sequence of at least one of a first complementary strand and a second complementary strand of individual double-stranded polynucleotides by adding one or more predetermined nucleotides in a base by base manner to the 3′ end of at least one of the first complementary strand and the second complementary strand;
- (b) circularizing a plurality of first complementary strands and a plurality of second complementary strands to form a plurality of single stranded polynucleotide circles, the one or more predetermined nucleotides defining a boundary between 3′ sequences and 5′ sequences of each single stranded polynucleotide circle;
- (c) amplifying by rolling circle amplification the single stranded polynucleotide circles to form a plurality of concatemers;
- (d) sequencing a plurality of first complementary strands and a plurality of second complementary strands, or amplification products thereof, to yield a plurality of sequencing reads;
- (e) identifying from the plurality of sequencing reads, a given first complementary strand and a given second complementary strand as originating from a common double-stranded polynucleotide based on (i) sequences of the respective 3′ end and 5′ ends adjacent to the one or more predetermined nucleotides, and (ii) sequences of the one or more predetermined nucleotides;
- (f) comparing polynucleotide sequences of the given first complementary strand and the given second complementary strand originating from the common double-stranded polynucleotide;
- and
- (g) calling a sequence difference in the given first complementary strand relative to a reference sequence as the sequence variant only when the given second complementary strand originating from the common double-stranded polynucleotide comprises a complement of the sequence difference.
Dependencies are inferred from the claim text.