Application of Immobilized Enzymes for Nanopore Library Construction

Abstract

The present disclosure relates, according to some embodiments, to methods for preparing a library for sequencing. For example, a method may comprise (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and/or separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase. In some embodiments, a tailing enzyme and/or a ligase used in library preparation may be immobilized enzymes.

What is claimed is: 
     
         1 . A method of preparing a library for sequencing, comprising:
 (a) in a coupled reaction, (i) contacting a population of nucleic acid fragments with a tailing enzyme to produce tailed fragments, and (ii) ligating to the tailed fragments a sequencing adapter with a ligase to produce adapter-tagged fragments; and   (b) separating adapter-tagged fragments from the tailing enzyme and the ligase to produce separated adapter-tagged fragments and, optionally, separated tailing enzyme and/or separated ligase.   
     
     
         2 . A method according to  claim 1 , wherein the tailing enzyme and the ligase are immobilized enzymes. 
     
     
         3 . A method according to  claim 1 , wherein the tailing enzyme is immobilized on a magnetic bead. 
     
     
         4 . A method according to  claim 3 , wherein the separating the adapter tagged fragments further comprises subjecting the coupled reaction to a magnetic field. 
     
     
         5 . A method according to  claim 1 , wherein the ligase is immobilized on a magnetic bead. 
     
     
         6 . A method according to  claim 5 , wherein the separating the adapter tagged fragments further comprises subjecting the coupled reaction to a magnetic field. 
     
     
         7 . A method according to  claim 1 , wherein the tailing enzyme and the ligase are immobilized on separate supports. 
     
     
         8 . A method according to  claim 1 , wherein the coupled reaction steps occur in a single tube, well, capillary, flow cell or surface. 
     
     
         9 . A method according to  claim 1 , wherein the tailing enzyme and the ligase are soluble enzymes. 
     
     
         10 . A method according to  claim 1 , wherein the population of nucleic acid fragments comprise ribonucleic acid fragments. 
     
     
         11 . A method according to  claim 1 , wherein the population of nucleic acid fragments comprise deoxyribonucleic acid fragments. 
     
     
         12 . A method according to  claim 1 , wherein the population of nucleic acid fragments has less than 100 ng of nucleic acids. 
     
     
         13 . A method according to  claim 1 , wherein the population of nucleic acid fragments has less than 10 ng of nucleic acids. 
     
     
         14 . A method according to  claim 1 , further comprising:
 (c) in a second coupled reaction, (i) contacting a second population of nucleic acid fragments with the separated tailing enzyme to produce additional tailed fragments, and (ii) ligating to the additional tailed fragments a second sequencing adapter with the separated ligase to produce additional adapter-tagged fragments, and   (d) separating the additional adapter-tagged fragments from the separated tailing enzyme and the separated ligase to produce separated additional adapter-tagged fragments, separated tailing enzyme, and separated ligase.   
     
     
         15 . A method according to  claim 14 , further comprising:
 (e) translocating the separated adapter-tagged fragments through one or more transmembrane pores;   (f) detecting electrical changes as the one or more separated adapter-tagged fragments are translocated through the one or more transmembrane pores in an insulating membrane to produce an electrical signal; and   (g) analyzing the electrical signal to generate a sequence read.   
     
     
         16 . A method according to  claim 15 , wherein the one or more transmembrane pores retain about 90% of their initial activity after two hours. 
     
     
         17 . A method according to  claim 15 , wherein the one or more transmembrane pores retain about 50% of their initial activity after 8 hours. 
     
     
         18 . A method according to  claim 15 , wherein the one or more transmembrane pores produce at least 900 sequence reads per transmembrane pore. 
     
     
         19 . A method according to  claim 1 , wherein the sequencing adapter is a single stranded adapter comprising:
 a leader sequence; and   a first sequence and a second sequence, wherein the first and second sequences are complementary to each other and define a hairpin,   
       wherein the leader sequence is configured to thread into the one or more transmembrane pores.