Methods and systems for nucleic acid sequencing

Abstract

The present disclosure provides methods and systems for processing a nucleotide mixture. A nucleotide mixture can be purified. A nucleotide mixture can be processed for use in nucleic acid synthesis. A nucleotide mixture can be processed for use in nucleic acid sequencing.

1 .- 125 . (canceled) 
     
     
         126 . A method for processing a mixture, comprising:
 (a) selecting from a set of canonical types of nucleotides or nucleotides analogs a subset of canonical types of nucleotides or nucleotide analogs;   (b) directing a mixture comprising a plurality of nucleotides or nucleotide analogs to a reaction space comprising a support having a plurality of nucleic acid molecules immobilized thereto, wherein a percentage of nucleotides or nucleotide analogs corresponding to said subset relative to all other nucleotides or nucleotide analogs in said mixture is greater than 50%; and   (c) incorporating nucleotides or nucleotide analogs that do not correspond to said subset from said mixture into said plurality of nucleic acid molecules such that said percentage is increased following said incorporating;   wherein (a)-(c) are performed in absence of sequencing or sequence identification of said plurality of nucleic acid molecules.   
     
     
         127 . The method of  claim 126  wherein, in (b), said percentage is greater than 80%, greater than 90%, greater than 95%, greater than 99%, greater than 99.9%, or greater than 99.99%. 
     
     
         128 . The method of  claim 126 , wherein said (c) is performed using a nucleic acid polymerizing enzyme comprising phi29 polymerase, Bst 3.0 polymerase, or a variant thereof. 
     
     
         129 . The method of  claim 126 , wherein said reaction space is further replenished with a plurality of nucleic acid polymerizing enzymes. 
     
     
         130 . A method for processing a mixture, comprising:
 (a) selecting from a set of canonical types of nucleotides or nucleotides analogs a subset of canonical types of nucleotides or nucleotide analogs;   (b) directing a mixture comprising a plurality of nucleotides or nucleotide analogs to a reaction space comprising a support having a plurality of nucleic acid molecules immobilized thereto, wherein a percentage of nucleotides or nucleotide analogs corresponding to said subset relative to all other nucleotides or nucleotide analogs in said mixture is less than 20%; and   (c) incorporating said nucleotides or nucleotide analogs corresponding to said subset from said mixture into said plurality of nucleic acid molecules such that said percentage is reduced following incorporation,   wherein (a)-(c) are performed in absence of sequencing or sequence identification of said plurality of nucleic acid molecules.   
     
     
         131 . The method of  claim 130 , wherein, in (b), said percentage is less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01%. 
     
     
         132 . The method of  claim 130 , wherein said plurality of nucleotides or nucleotide analogs includes deoxynucleotides or dideoxynucleotides. 
     
     
         133 . The method of  claim 132 , wherein said plurality of nucleotides or nucleotide analogs comprises nucleotides or nucleotide analogs selected from the group consisting of deoxyadenosine triphosphate (dATP), 2′,3′-ideoxyadenosine-5′triphosphate (ddATP), deoxyguanosine triphosphate (dGTP), 2′,3′-dideoxyguanosine-5′-triphosphate (ddGTP), deoxycytidine triphosphate (dCTP), 2′,3′-dideoxycytidine-5′-triphosphate (ddCTP), deoxythymidine triphosphate (dTTP), 2′,3′-dideoxythymidine-5′-triphosphate (ddTTP), deoxyuridine triphosphate (dUTP), 2′,3′-dideoxyuridine-5′-triphosphate (ddUTP), and a variant thereof. 
     
     
         134 . The method of  claim 130 , wherein said incorporating in (c) yields double stranded nucleic acid molecules from said plurality of nucleic acid molecules comprising said nucleotides or nucleotide analogs corresponding to said subset, and wherein, subsequent to (c), said reaction space is replenished with an additional plurality of nucleic acid molecules, which additional plurality of nucleic acid molecules does not include said nucleotides or nucleotide analogs corresponding to said subset incorporated in (c). 
     
     
         135 . The method of  claim 134 , wherein said reaction space is replenished by directing an additional support immobilized with said additional plurality of nucleic acid molecules to said reaction space. 
     
     
         136 . The method of  claim 135 , wherein said support is a polymer matrix, a particle, or a fiberglass structure. 
     
     
         137 . The method of  claim 136 , wherein said particle is a bead or a gel particle. 
     
     
         138 . The method of  claim 137 , wherein said particle is said gel particle, wherein said gel particle is an aerogel particle or a hydrogel particle. 
     
     
         139 . The method of  claim 134 , wherein said reaction space is replenished by denaturation of said double stranded nucleic acid molecules and removal of resultant single stranded nucleic acid molecules that are not immobilized to said support. 
     
     
         140 . The method of  claim 139 , wherein (1) said denaturation is chemical denaturation or thermal denaturation; or (2) wherein said removal of said resultant single stranded nucleic acid molecules comprises washing, filtering, or a combination thereof. 
     
     
         141 . A method for synthesizing a nucleic acid molecule, comprising:
 (a) directing a plurality of nucleotides or nucleotide analogs to a reaction space comprising a support having a plurality of nucleic acid molecules immobilized thereto;   (b) incorporating a subset of nucleotides or nucleotide analogs from said plurality of nucleotides or nucleotide analogs into said plurality of nucleic acid molecules, thereby providing a remainder of said plurality of nucleotides or nucleotides analogs; and   (c) using said remainder of said plurality of nucleotides or nucleotide analogs to perform nucleic acid synthesis, thereby synthesizing said nucleic acid molecule;   wherein (a)-(b) are performed in absence of sequencing or sequence identification of said plurality of nucleic acid molecules.   
     
     
         142 . The method of  claim 141 , wherein said subset consists of nucleotides or nucleotide analogs of one, two, or three canonical types. 
     
     
         143 . The method of  claim 141 , wherein said plurality of nucleic acid molecules contains nucleotides complementary to said subset of nucleotides or nucleotide analogs. 
     
     
         144 . The method of  claim 141 , wherein said plurality of nucleic acid molecules contains a greater number of nucleotides complementary to said subset of nucleotides or nucleotide analogs relative to all other nucleotides. 
     
     
         145 . The method of  claim 141 , wherein said plurality of nucleic acid molecules does not contain nucleotides that are not complementary to said subset of nucleotides or nucleotide analogs.