Fast-forward sequencing by synthesis methods

Abstract

Described herein are methods of generating a coupled sequencing read pair for a polynucleotide, and methods of analyzing the coupled sequencing read pair. The coupled sequencing read pair can be analyzed to detect polynucleotide variants, including at loci that are not directly sequenced within the coupled sequencing read pair. Other analytical methods can include using coupled sequencing read pairs to construct or validate a consensus sequence. The coupled sequencing read pair may be generated for a polynucleotide by generating sequencing data for a first region by extending a primer using labeled nucleotides; further extending the primer through a second region using nucleotides provided in a second region flow order, wherein primer extension through the second region is faster than primer extension through the first region; and generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer using labeled nucleotides.

1 . A method of generating a coupled sequencing read pair from a polynucleotide, comprising:
 (a) hybridizing the polynucleotide to a primer to form a hybridized template;   (b) generating sequencing data associated with a sequence of a first region of the polynucleotide by extending the primer using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide;   (c) further extending the primer extended in step (b) through a second region using nucleotides provided in a second region flow order, wherein (i) the primer is extended through the second region without detecting the presence or absence of a label of a nucleotide incorporated into the extending primer, (ii) a mixture of at least two different types of nucleotide bases are used in at least one step of the second region flow order, or (iii) extension of the primer through the second region proceeds faster than the extension of the primer in step (b); and   (d) generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer extended in step (c) using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide.   
     
     
         2 . The method of  claim 1 , wherein extension of the primer through the second region proceeds faster than the extension of the primer through the first region. 
     
     
         3 . The method of  claim 1 , further comprising associating the sequencing data of the first region with the sequencing data of the third region. 
     
     
         4 . A method of generating a coupled sequencing read pair from a polynucleotide, comprising:
 (a) hybridizing a primer to a first region of the polynucleotide to form a hybridized template;   (b) extending the primer through a second region using nucleotides provided in a second region flow order, wherein (i) the primer is extended through the second region without detecting the presence or absence of a label of a nucleotide incorporated into the extending primer, or (ii) a mixture of at least two different types of nucleotide bases are used in at least one step of the second region flow order; and   (c) generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer extended in step (b) using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide.   
     
     
         5 . The method of  claim 4 , wherein the first region comprises a naturally occurring sequence targeted by the primer. 
     
     
         6 . The method of  claim 1 , wherein the primer is extended through the second region without detecting the presence or absence of a label of a nucleotide incorporated into the extending primer. 
     
     
         7 . The method of  claim 1 , wherein at least a portion of the nucleotides used to extend the primer through the second region are unlabeled nucleotides. 
     
     
         8 . The method of  claim 1 , wherein the nucleotides used to extend the primer through the second region are unlabeled nucleotides. 
     
     
         9 . The method of  claim 1 , wherein a mixture of at least two different types of nucleotide bases are used in at least one step of the second region flow order. 
     
     
         10 . The method of  claim 1 , wherein the second region flow order comprises five or more nucleotide flows. 
     
     
         11 . The method of  claim 10 , wherein each of the nucleotide flows comprises a single nucleotide base. 
     
     
         12 . The method of  claim 10 , wherein the second region flow order induces a signal change at more than two flow positions for 50% or more of possible SNP permutations at 5% or more of random sequencing start positions. 
     
     
         13 . The method of  claim 10 , wherein the second region flow order has an efficiency of 0.6 or more base incorporations per flow. 
     
     
         14 . The method of  claim 1 , further comprising determining expected sequencing data for the second region using a reference sequence and the second region flow order. 
     
     
         15 . The method of  claim 1 , wherein the primer is extended through the third region using nucleotides provided in a third region flow order, the method further comprising determining expected sequencing data for the third region using a reference sequence for the second region, the second region flow order, the third region flow order, and a reference sequence for the third region. 
     
     
         16 . The method of  claim 15 , wherein the third region flow order comprises five or more nucleotide flows. 
     
     
         17 . The method of  claim 16 , wherein each of the nucleotide flows comprises a single nucleotide base. 
     
     
         18 . The method of  claim 16 , wherein the third region flow order induces a signal change at more than two flow positions for 50% or more of possible SNP permutations at 5% or more of random sequencing start positions. 
     
     
         19 . The method of  claim 16 , wherein the third region flow order has an efficiency of 0.6 or more base incorporations per flow. 
     
     
         20 . The method of  claim 1 , wherein the primer is extended through the third region using nucleotides provided in a third region flow order, the method further comprising determining expected sequencing data for the third region using a reference sequence for the second region, the second region flow order, the third region flow order, and sequencing data associated with the sequence of the third region, wherein the sequencing data associated with the sequence of the third region is the same or different sequencing data generated for the third region. 
     
     
         21 . The method of  claim 14 , wherein the expected reference data for the second region or the third region comprises a binary or non-binary flowgram. 
     
     
         22 . The method of  claim 14 , further comprising determining expected test variant sequencing data for the second region using the second region flow order and a second reference sequence for the second region, wherein the second reference sequence comprises the test variant. 
     
     
         23 . The method of  claim 22 , wherein the primer is extended through the third region using nucleotides provided in a third region flow order, the method further comprising determining expected test variant sequencing data for the third region using the second reference sequence for the second region, the second region flow order, the third region flow order, and a reference sequence for the third region. 
     
     
         24 . The method of  claim 22 , wherein the primer is extended through the third region using nucleotides provided in a third region flow order, the method further comprising determining expected test variant sequencing data for the third region using the second reference sequence for the second region, the second region flow order, the third region flow order, and sequencing data associated with the sequence of the third region, wherein the sequencing data associated with the sequence of the third region is the same or different sequencing data generated for the third region. 
     
     
         25 . The method of  claim 22 , wherein the expected reference sequencing data for the second region or the third region comprises a binary or non-binary flowgram. 
     
     
         26 . A method of mapping a coupled sequencing read pair to a reference sequence, comprising:
 mapping a first region or portion thereof, or a third region or portion thereof, of a coupled sequencing read pair generated according to the method of  claim 1  to a reference sequence; and   mapping the unmapped first region or portion thereof, or the unmapped third region or portion thereof, to the reference sequence using distance information indicative of the length of the second region.   
     
     
         27 . A method of detecting a structural variant, comprising:
 mapping a first region or portion thereof, or a third region or portion thereof, of a coupled sequencing read pair generated according to the method of  claim 1 , to a reference sequence;   determining an expected locus within a reference sequence for the unmapped first region or portion thereof, or the unmapped third region or portion thereof, using distance information indicative of the length of the second region;   determining expected sequencing data for a sequence at the expected locus based on the reference sequence; and   detecting the structural variant by comparing the sequencing data of the unmapped first region or portion thereof, or the unmapped third region or portion thereof, to the expected sequencing data, wherein a difference between the sequencing data of the unmapped first region or portion thereof, or the unmapped third region or portion thereof, and the expected sequencing data indicates the structural variant.   
     
     
         28 . A method of detecting a structural variant, comprising:
 mapping a first region or portion thereof or a third region or portion thereof, of a coupled sequencing read pair generated according to the method of  claim 1  to a reference sequence, wherein the unmapped first region, or the unmapped third region, is unmappable within the reference sequence.   
     
     
         29 . A method of detecting a structural variant, comprising:
 mapping a first region or portion thereof and a third region or portion thereof, of a coupled sequencing read pair generated according to the method of  claim 1  to a reference sequence;   determining a mapped distance information between the mapped first region and the mapped third region; and   detecting the structural variant by comparing the mapped distance information to an expected distance information of the second region, wherein a difference between the mapped distance information and the expected distance information indicates the structural variant.   
     
     
         30 - 31 . (canceled) 
     
     
         32 . A method of mapping a coupled sequencing read pair to a reference sequence, comprising:
 mapping a first region or portion thereof and a third region or portion thereof of a coupled sequencing read pair generated according to the method of  claim 1  to a reference sequence at two or more different position pairs comprising a first position and a second position; and   selecting a correct position pair using first distance information indicative of the length of the second region and second distance information indicative of the distances between the first position and the second position for the two or more position pairs.   
     
     
         33 . A method of detecting a variant between two sequenced regions of a coupled sequencing read pair generated according to the method of  claim 1 , wherein the primer extended is extended through the third region using nucleotides provided in a third region flow order, comprising:
 mapping the first region or portion thereof to a reference sequence;   determining expected sequencing data for the third region or portion thereof using (1) a reference sequence for the second region, the second region flow order, the third region flow order, and a reference sequence for the third region, or (2) a reference sequence for the second region, the second region flow order, the third region flow order, and generated sequencing data associated with the sequence of the third region, wherein the generated sequence data associated with the sequence of the third region is the same or different sequence data generated for the third region; and   detecting the presence of a variant by comparing the expected sequencing data for the third region to the generated sequencing data associated with the sequence of the third region.   
     
     
         34 - 39 . (canceled) 
     
     
         40 . A method of generating a coupled sequencing read pair for detecting the presence of a base transversion in an unsequenced region of a polynucleotide, comprising:
 (a) hybridizing the polynucleotide to a primer to form a hybridized template;   (b) generating sequencing data associated with a sequence of a first region of the polynucleotide by extending the primer using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide;   (c) further extending the primer extended in step (b) through a second region using a flow order comprising alternating nucleotide pairs of (1) cytosine and thymine, and (2) adenine and guanine; and   (d) generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer extended in step (c) using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide.   
     
     
         41 . A method of generating a coupled sequencing read pair from a polynucleotide, comprising:
 (a) hybridizing a primer to a first region of the polynucleotide to form a hybridized template;   (b) extending the primer through a second region using a flow order comprising alternating nucleotide pairs of (1) cytosine and thymine, and (2) adenine and guanine; and   (c) generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer extended in step (b) using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide.   
     
     
         42 - 43 . (canceled) 
     
     
         44 . A method of detecting the presence of a base transversion in an unsequenced region of a polynucleotide, comprising:
 mapping a first region or portion thereof, and a third region or a portion thereof, of a coupled sequencing read pair generated according to the method of  claim 41 , wherein the primer is extended through the third region using nucleotides provided in a third region flow order, to a reference sequence;   determining expected sequencing data for the third region using the second region flow order, the third region flow order, and the reference sequence; and   detecting the presence of the base transversion based on the difference between expected sequencing data for the third region and the generated sequencing data for the third region.   
     
     
         45 - 47 . (canceled) 
     
     
         48 . A method of generating one or more consensus sequences, comprising assembling a plurality of coupled sequencing read pairs generated according to the method of  claim 1 . 
     
     
         49 . (canceled) 
     
     
         50 . A method of validating a status of a test variant, comprising:
 comparing a status of the variant across a plurality of overlapping coupled sequencing read pairs generated according to the method of  claim 1 , the plurality of overlapping coupled sequencing read pairs comprising a locus corresponding to a locus of the test variant;   validating the status of the variant of based on the comparison.   
     
     
         51 . A method for detecting a short genetic variant in a test sample, comprising:
 generating a coupled sequencing read pair according to the method of  claim 1 ;   comparing the sequencing data associated with a sequence of third region of the polynucleotide to expected sequencing data for an expected sequence of the third region of the polynucleotide; and   calling the presence or absence of the short genetic variant in the second region of the polynucleotide.   
     
     
         52 - 54 . (canceled) 
     
     
         55 . The method of  claim 1 , wherein generating the coupled sequencing read pair further comprises:
 further extending the primer through a fourth region using nucleotides provided in a fourth region flow order, wherein (i) the primer is extended through the fourth region without detecting the presence or absence of a label of a nucleotide incorporated into the extending primer, (ii) a mixture of at least two different types of nucleotide bases are used in at least one step of the fourth region flow order, or (iii) extension of the primer through the fourth region proceeds faster than the extension of the primer through the first region or the third region; and   generating sequencing data associated with a sequence of a fifth region of the polynucleotide by further extending the primer extended through the fourth using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide.   
     
     
         56 - 57 . (canceled) 
     
     
         58 . A method of detecting a short genetic variant in a test sample, comprising:
 (a) amplifying a polynucleotide using rolling circle amplification (RCA) to generate a RCA-amplified polynucleotide comprising at least a first copy of the polynucleotide and a second copy of the polynucleotide;   (b) hybridizing the RCA-amplified polynucleotide to a primer to form a hybridized template;   (c) generating sequencing data associated with a sequence of a first region of the polynucleotide within the first copy of the polynucleotide by extending the primer using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide;   (d) further extending the primer through a second region of the polynucleotide within the first copy of the polynucleotide using nucleotides provided in a second region flow order, wherein (i) the primer is extended through the second region of the polynucleotide within the first copy of the polynucleotide without detecting the presence or absence of a label of a nucleotide incorporated into the extending primer, (ii) a mixture of at least two different types of nucleotide bases are used in at least one step of the second region flow order, or (iii) extension of the primer through the second region of the polynucleotide within the first copy of the polynucleotide proceeds faster than the extension of the primer through the first region;   (e) generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide;   (f) comparing the sequencing data generated for the third region of the polynucleotide to expected sequencing data for an expected sequence of the third region of the polynucleotide;   (g) calling the presence of the short genetic variant in the second region of the polynucleotide;   (h) generating sequencing data associated with a sequence of the second region of the polynucleotide within the second copy of the polynucleotide by extending the primer using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide; and   (i) calling the identity of the short genetic variant in the second region of the polynucleotide.   
     
     
         59 . (canceled) 
     
     
         60 . A method of detecting a short genetic variant in a test sample, comprising:
 (a) amplifying a polynucleotide using rolling circle amplification (RCA) to generate a RCA-amplified polynucleotide comprising at least a first copy of the polynucleotide and a second copy of the polynucleotide;   (b) hybridizing a primer to a first region of the polynucleotide within the first copy of the polynucleotide to form a hybridized template;   (c) extending the primer through a second region of the polynucleotide within the first copy of the polynucleotide using nucleotides provided in a second region flow order, wherein (i) the primer is extended through the second region of the polynucleotide within the first copy of the polynucleotide without detecting the presence or absence of a label of a nucleotide incorporated into the extending primer, or (ii) a mixture of at least two different types of nucleotide bases are used in at least one step of the second region flow order;   (d) generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide;   (e) comparing the sequencing data generated for the third region of the polynucleotide to expected sequencing data for an expected sequence of the third region of the polynucleotide;   (f) calling the presence of the short genetic variant in the second region of the polynucleotide;   (g) generating sequencing data associated with a sequence of the second region of the polynucleotide within the second copy of the polynucleotide by extending the primer using labeled nucleotides, and detecting the presence or absence of an incorporated labeled nucleotide; and   (h) calling the identity of the short genetic variant in the second region of the polynucleotide.   
     
     
         61 - 65 . (canceled) 
     
     
         66 . A method of synchronizing sequencing primers within a sequencing cluster, comprising:
 (a) hybridizing primers to polynucleotide copies within a sequencing cluster;   (b) extending the primers through a first region of the polynucleotide copies using labeled nucleotides according to a first region flow cycle;   (c) extending the primers through a second region of the polynucleotide copies using one or more re phasing flows, wherein a mixture of at least two different types of nucleotide bases are used in each of the one or more re-phasing flows; and   (d) extending the primers through a third region of the polynucleotide copies using labeled nucleotides according to a third region flow cycle.   
     
     
         67 - 71 . (canceled)