Methods for sequencing with single frequency detection

Abstract

The present disclosure provides methods and systems for sequencing nucleic acid molecules using a single frequency during detection, or fewer frequencies than types of nucleotide bases identified during detection. Methods and systems of the present disclosure may involve transiently binding nucleotides. Methods and systems provided herein may enable sequences to be determined at a higher accuracy and efficiency.

1 .- 34 . (canceled) 
     
     
         35 . A method for nucleic acid sequencing, comprising:
 (a) providing a substrate comprising at least a first set of nucleic acid molecules and a second set of nucleic acid molecules, wherein said first set of nucleic acid molecules has sequence homology to a first template, and said second set of nucleic acid molecules has sequence homology to a second template, wherein said first template is different than said second template;   (b) bringing said first set of nucleic acid molecules and said second set of nucleic acid molecules in contact with a reaction mixture comprising nucleotides of at least two different types comprising an adenine base type, under conditions sufficient to permit transient binding of at least a subset of said nucleotides to nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules;   (c) detecting a first optical signal from said first set of nucleic acid molecules and a second signal from said second set of nucleic acid molecules, wherein said first optical signal and said second optical signal have a first frequency, and wherein said first optical signal and said second optical signal have different intensities; and   (d) using intensities of said first optical signal and said second optical signal to identify types of nucleotides of said at least said subset of said nucleotides transiently bound to said nucleic acid molecules of said first set of nucleic acid molecules and said nucleic acid molecules of said second set of nucleic acid molecules, thereby sequencing said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules.   
     
     
         36 . The method of  claim 35 , wherein said conditions sufficient to permit said transient binding comprise a presence of a metal cation, and wherein said metal cation is selected from the group consisting of calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium. 
     
     
         37 . The method of  claim 35 , further comprising, subsequent to (d), removing said at least said subset of said nucleotides transiently bound to said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules. 
     
     
         38 . The method of  claim 37 , further comprising heating said substrate to remove said at least said subset of said nucleotides transiently bound to said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules. 
     
     
         39 . The method of  claim 37 , further comprising bringing said first set of nucleic acid molecules and said second set of nucleic acid molecules in contact with an additional reaction mixture comprising additional nucleotides of at least two types, under conditions sufficient to permit non-transient binding of at least a subset of said additional nucleotides from said additional reaction mixture to said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules. 
     
     
         40 . The method of  claim 35 , wherein said reaction mixture comprises nucleotides of at least three different types. 
     
     
         41 . A method for nucleic acid sequencing, comprising:
 (a) providing a substrate comprising at least a first set of nucleic acid molecules and a second set of nucleic acid molecules, wherein said first set of nucleic acid molecules has sequence homology to a first template, and said second set of nucleic acid molecules has sequence homology to a second template, wherein said first template is different than said second template;   (b) bringing said first set of nucleic acid molecules and said second set of nucleic acid molecules in contact with a reaction mixture comprising nucleotides of at least two different types comprising a thymine base type, under conditions sufficient to permit non-transient binding of at least a subset of said nucleotides to nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules, wherein said nucleotides comprise a reversible terminator;   (c) detecting a first optical signal from said first set of nucleic acid molecules and a second signal from said second set of nucleic acid molecules, wherein said first optical signal and said second optical signal have a first frequency, and wherein said first optical signal and said second optical signal have different intensities; and   (d) using intensities of said first optical signal and said second optical signal to identify types of nucleotides of said at least said subset of said nucleotides non-transiently bound to said nucleic acid molecules of said first set of nucleic acid molecules and said nucleic acid molecules of said second set of nucleic acid molecules, thereby sequencing said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules.   
     
     
         42 . The method of  claim 41 , wherein said conditions sufficient to permit said non-transient binding comprise a presence of a metal cation, and wherein said metal cation is selected from the group consisting of calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium. 
     
     
         43 . The method of  claim 41 , further comprising, subsequent to (d), removing a blocking group from said subset of said nucleotides non-transiently bound to said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules. 
     
     
         44 . The method of  claim 41 , further comprising bringing said first set of nucleic acid molecules and said second set of nucleic acid molecules in contact with an additional reaction mixture comprising additional nucleotides of at least two types, under conditions sufficient to permit transient binding of at least a subset of said additional nucleotides from said additional reaction mixture to said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules. 
     
     
         45 . The method of  claim 44 , wherein said conditions sufficient to permit said transient binding comprise a presence of a second metal cation, and wherein second metal cation is selected from the group consisting of calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium. 
     
     
         46 . The method of  claim 41 , further comprising bringing said first set of nucleic acid molecules and said second set of nucleic acid molecules in contact with an additional reaction mixture comprising additional nucleotides of at least two types, under second conditions sufficient to permit incorporation of at least a subset of said additional nucleotides from said additional reaction mixture to said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules. 
     
     
         47 . The method of  claim 46 , wherein said conditions sufficient to permit said non-transient binding and said second conditions sufficient to permit said non-transient binding are same conditions. 
     
     
         48 . The method of  claim 46 , wherein said conditions sufficient to permit said non-transient binding and said second conditions sufficient to permit said non-transient binding are different conditions. 
     
     
         49 . The method of  claim 41 , wherein said reaction mixture comprises nucleotides of at least three different types. 
     
     
         50 . A method for nucleic acid sequencing, comprising:
 (a) providing a substrate comprising at least a first set of nucleic acid molecules and a second set of nucleic acid molecules, wherein said first set of nucleic acid molecules has sequence homology to a first template, and said second set of nucleic acid molecules has sequence homology to a second template, wherein said first template is different than said second template;   (b) bringing said first set of nucleic acid molecules and said second set of nucleic acid molecules in contact with a reaction mixture comprising nucleotides of at least two different types comprising a cytosine base type, under conditions sufficient to permit non-transient binding of at least a subset of said nucleotides to at least a first subset of nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules, wherein said nucleotides are unterminated;   (c) detecting a first optical signal from said first set of nucleic acid molecules and a second signal from said second set of nucleic acid molecules, wherein said first optical signal and said second optical signal have a first frequency, and wherein said first optical signal and said second optical signal have different intensities; and   (d) using intensities of said first optical signal and said second optical signal to identify types of nucleotides of said at least said subset of said nucleotides non-transiently bound to said nucleic acid molecules of said first set of nucleic acid molecules and said nucleic acid molecules of said second set of nucleic acid molecules, thereby sequencing said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules.   
     
     
         51 . The method of  claim 50 , wherein said conditions sufficient to permit said non-transient binding comprise a presence of a metal cation, and wherein said metal cation is selected from the group consisting of calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium. 
     
     
         52 . The method of  claim 50 , wherein said reaction mixture comprises nucleotides of at least three different types. 
     
     
         53 . The method of  claim 50 , wherein said reaction mixture comprises nucleotides of at least four different types. 
     
     
         54 . The method of  claim 50 , further comprising, subsequent to (d), bringing said first set of nucleic acid molecules and said second set of nucleic acid molecules in contact with an additional reaction mixture comprising additional nucleotides of at least two different types, under second conditions sufficient to permit non-transient binding of at least a subset of said additional nucleotides from said additional mixture to at least a second subset of said nucleic acid molecules of said first set of nucleic acid molecules and said second set of nucleic acid molecules, and wherein said second conditions sufficient to permit said non-transient binding comprise a presence of a second metal cation, and wherein said second metal cation is selected from the group consisting of calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, and strontium.