Methods of sequencing nucleic acid molecules
The present disclosure provides methods for nucleic acid sequence identification. The methods may comprise bringing a plurality of nucleic acid molecules in contact with a reaction mixture including a concentration of nucleotides that results in fractional labeling of the nucleic acid molecules. The methods may comprise starting a next reversibly-terminated, sequencing cycle prior to completion of unblocking of reversible terminators in a previous sequencing cycle.
1 . A method for sequencing, comprising:
(a) providing a plurality of nucleic acid molecules, wherein said plurality of nucleic acid molecules has sequence identity with respect to one another;
(b) repeating, at least four times, the following steps:
(i) contacting said plurality of nucleic acid molecules with a first reaction solution comprising a first plurality of nucleotides at a first concentration, under conditions sufficient to incorporate said first plurality of nucleotides into a first plurality of growing strands of a first subset of said plurality of nucleic acid molecules by a first polymerase in a first primer extension reaction, wherein said first plurality of nucleotides comprises only one kind of identical purine or pyrimidine nucleotides and comprises labeled nucleotides that comprise a first detectable label;
(ii) subsequent to step (i), removing nucleotides of said first reaction solution that are not incorporated into the first plurality of growing strands of the first subset of said plurality of nucleic acid molecules by bringing a mixture formed in step (i) into contact with a first washing solution;
(iii) subsequent to step (ii), bringing a mixture formed in step (ii) into contact with a second reaction solution comprising a second plurality of nucleotides at a second concentration, under conditions sufficient to incorporate said second plurality of nucleotides into a second plurality of growing strands of a second subset of said plurality of nucleic acid molecules by a second polymerase in a second primer extension reaction, wherein said second concentration is higher than said first concentration, wherein said second plurality of nucleotides comprises only said one kind of identical purine or pyrimidine nucleotides and comprises only unlabeled nucleotides;
(iv) subsequent to step (iii), removing nucleotides of said second reaction solution that are not incorporated into the second plurality of growing strands of the second subset of said plurality of nucleic acid molecules by bringing a mixture formed in step (iii) into contact with a second washing solution; and
(v) detecting one or more signals from said first detectable label in the mixture formed in step (ii) or a mixture formed in step (iv), wherein the one or more signals are indicative that said first plurality of nucleotides is incorporated into the first plurality of growing strands of the first subset of said plurality of nucleic acid molecules, wherein, when step (b) is repeated for at least four times, in each time of the at least four times, said one kind of identical purine or pyrimidine nucleotides in step (b) is different.
2 . The method of claim 1 , wherein said conditions sufficient to incorporate said first plurality of nucleotides into said first plurality of growing strands of said first subset of said plurality of nucleic acid molecules are different from said conditions sufficient to incorporate said second plurality of nucleotides into said second plurality of growing strands of said second subset of said plurality of nucleic acid molecules.
3 . The method of claim 1 , wherein said first plurality of nucleotides further comprises unlabeled nucleotides.
4 . The method of claim 3 , wherein the concentration of said unlabeled nucleotides of said first plurality of nucleotides is higher than the concentration of said labeled nucleotides of said first plurality of nucleotides.
5 . The method of claim 1 , wherein said first plurality of nucleotides or said second plurality of nucleotides is non-terminated nucleotides.
6 . The method of claim 1 , wherein said first plurality of nucleotides and said second plurality of nucleotides are non-terminated nucleotides.
7 . The method of claim 1 , wherein said plurality of nucleic acid molecules comprises a homopolymer sequence, wherein: (1) a subset of said first plurality of nucleotides is incorporated into nucleotide positions of a growing strand of said first plurality of growing strands of said first subset of said plurality of nucleic acid molecules which is complementary to said homopolymer sequence or (2) a subset of said second plurality of nucleotides is incorporated into nucleotide positions in a growing strand of said second plurality of growing strands of said second subset of said plurality of nucleic acid molecules which is complementary to said homopolymer sequence.
8 . The method of claim 1 , wherein said first plurality of growing strands and said second plurality of growing strands do not share a common growing strand.
9 . The method of claim 1 , wherein said first plurality of growing strands and said second plurality of growing strands share at least one common growing strand.
10 . The method of claim 1 , further comprising, subsequent to step (v), bringing a mixture formed in step (v) into contact with a cleaving solution to cleave the first detectable label from said first plurality of growing strands of said first subset of said plurality of nucleic acid molecules.
11 . The method of claim 1 , wherein nucleic acid molecules of at least a portion of said first subset of said plurality of nucleic acid molecules and nucleic acid molecules of at least a portion of said second subset of said plurality of nucleic acid molecules are identical.
12 . The method of claim 1 , further comprising, subsequent to step (iii), bringing said mixture formed in step (iii) into contact with a third reaction solution comprising a third plurality of nucleotides, under conditions sufficient to incorporate said third plurality of nucleotides into a third plurality of growing strands of a third subset of said plurality of nucleic acid molecules by a third polymerase in a third primer extension reaction, wherein said third plurality of nucleotides comprises only said one kind of identical purine or pyrimidine nucleotides.
13 . The method of claim 1 , further comprising, repeating steps (i)-(iv) using a third reaction solution instead of said first reaction solution, wherein said third reaction solution comprises a third plurality of nucleotides, wherein said third plurality of nucleotides comprises only another one kind of identical purine or pyrimidine nucleotides that is different from said one kind of identical purine or pyrimidine nucleotides and comprises labeled nucleotides that comprise a second detectable label, and using a fourth reaction solution instead of said second reaction solution, wherein said fourth reaction solution comprises a fourth plurality of nucleotides, wherein said fourth plurality of nucleotides comprises only said another one kind of identical purine or pyrimidine nucleotides and comprises only unlabeled nucleotides; and repeating step (v), wherein detecting one or more signals from said second detectable label in a mixture formed in the repeated step (ii) or a mixture formed in the repeated step (iv) indicates that said third plurality of nucleotides is incorporated into said plurality of nucleic acid molecules.
14 . The method of claim 1 , wherein step (iii) is performed prior to said detecting one or more signals from said first detectable label in said mixture formed in step (iv).
15 . The method of claim 1 , wherein said second concentration is at least about 10% higher than said first concentration.
16 . The method of claim 1 , wherein said second concentration is at least about 500% higher than said first concentration.
17 . The method of claim 1 , wherein said first plurality of nucleotides or said second plurality of nucleotides are reversibly terminated nucleotides.