Methods and Uses of Introducing Mutations into Genetic Material for Genome Assembly

Abstract

Methods of sequencing and assembling a nucleic acid sequence from a nucleic acid sample containing repetitive or low-information regions, which are typically difficult to sequence and/or assemble are provided. The methods of sequencing and assembling introduce mutations into the sample to increase sequence diversity between various repetitive regions present in the nucleic acid sample. This sequence diversity allows various segments to assemble independently of different, but similar sequences present in the nucleic acid sample.

1 .- 20 . (canceled) 
     
     
         21 . A method of assembling a genome sequence comprising:
 obtaining a nucleic acid sample;   mutating the nucleic acid sample;   sequencing the mutated nucleic acid sample; and   assembling the sequenced mutated nucleic acid sample to build a genome sequence.   
     
     
         22 . The method of  claim 21 , wherein the mutating is done by ionizing radiation. 
     
     
         23 . The method of  claim 22 , wherein the ionizing radiation is UV radiation, gamma radiation, alpha particles, beta particles, and/or combinations thereof. 
     
     
         24 . The method of  claim 21 , wherein the mutating is done by an amplification-based technique. 
     
     
         25 . The method of  claim 24 , wherein the amplification-based technique is polymerase chain reaction (PCR), multiple strand displacement amplification (MDA), and/or rolling circle amplification (RCA). 
     
     
         26 . The method of  claim 24 , wherein the amplification-based technique is polymerase chain reaction (PCR). 
     
     
         27 . The method of  claim 24 , wherein the amplification-based technique is multiple strand displacement amplification (MDA). 
     
     
         28 . The method of  claim 24 , wherein the amplification-based technique is rolling circle amplification (RCA). 
     
     
         29 . The method of  claim 25 , further comprising introducing mutations into the sample during the amplification by incorporating 2′-Deoxy-P-nucleoside-5′-Triphosphate (dPTP). 
     
     
         30 . The method of  claim 25 , wherein the polymerase exhibits strand displacement properties and a high level of processivity. 
     
     
         31 . The method of  claim 30 , wherein the polymerase is Phi29 DNA polymerase or an enzymatic variant thereof. 
     
     
         32 . The method of  claim 30 , wherein the polymerase is Phi29 DNA polymerase or EquiPhi29. 
     
     
         33 . The method of  claim 25 , further comprising introducing mutations into the sample during the amplification by incorporating 2′-Deoxy-P-nucleoside-5′-Triphosphate (dPTP). 
     
     
         34 . The method of  claim 25 , wherein the polymerase exhibits strand displacement properties and a high level of processivity. 
     
     
         35 . The method of  claim 30 , wherein the polymerase is Phi29 DNA polymerase or an enzymatic variant thereof. 
     
     
         36 . The method of  claim 30 , wherein the polymerase is Phi29 DNA polymerase or EquiPhi29. 
     
     
         37 . A method comprising:
 obtaining a sample comprising a template nucleic acid;   introducing mutations into the sample via amplification of the template nucleic acid to create a mutated sample, wherein the amplification comprises contacting the template nucleic acid with a polymerase under conditions that promote amplification of the template nucleic acid;   sequencing the amplified nucleic acids comprising the mutated sample; and   assembling the sequences of the amplified nucleic acids comprising the mutated sample to build a genome or genomic region sequence;   wherein the polymerase is Phi29 DNA polymerase or an enzymatic variant thereof; and   wherein introducing mutations into the sample comprises incorporating 2′-Deoxy-P-nucleoside-5′-Triphosphate (dPTP) during the amplification;   wherein introducing the mutations with the polymerase and the dPTP facilitates the assembly and lowers the rate of misassembly.   
     
     
         38 . The method of  claim 37 , wherein the enzymatic variant thereof is EquiPhi29. 
     
     
         39 . The method of  claim 37 , wherein the amplification is polymerase chain reaction (PCR), multiple strand displacement amplification (MDA), and/or rolling circle amplification (RCA). 
     
     
         40 . The method of  claim 38 , wherein the amplification is polymerase chain reaction (PCR), multiple strand displacement amplification (MDA), and/or rolling circle amplification (RCA).