Methods for assembling and reading nucleic acid sequences from mixed populations

Abstract

The disclosure relates to methods for obtaining nucleic acid sequence information by constructing a nucleic acid library and reconstructing longer nucleic acid sequences by assembling a series of shorter nucleic acid sequences.

1 . A method for sequencing a nucleic acid molecule, the method comprising:
 (a) providing a plurality of clonal nucleic acid molecules each comprising at least a first adapter having the same barcode sequence attached in proximity to a first end;   (b) fragmenting the clonal nucleic acid molecules adjacent to random portions of the nucleic acid molecules to provide a second end;   (c) joining the first end with the second end to provide a plurality of circularized nucleic acid molecules having the barcode sequence adjacent to the random portion of the nucleic acid molecule;   (d) fragmenting the plurality of circularized nucleic acid molecules from (c);   (e) sequencing the barcode and the random portions of the nucleic acid molecules; and   (f) assembling the sequences from the plurality of random portions of the nucleic acid molecules.   
     
     
         2 . The method of  claim 1 , wherein the method is performed with a plurality of clonal nucleic acid populations, each population having a different barcode sequence attached thereto, and a separate sequence is assembled in (f) for each of the barcode sequences. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 2 , comprising, prior to step (a):
 (i) providing a plurality of nucleic acid molecules;   (ii) providing a plurality of first adapters, each adapter comprising a first region that is identical for each of the adapters and a barcode sequence that is unique for each of the adapters;   (iii) attaching the adapters of (ii) to the target nucleic acid molecules of (i) to create a plurality of target molecules with adapters at both the 5′ and 3′ ends; and   (iv) amplifying the plurality of nucleic acid molecules molecules of (iii) to produce the plurality of clonal nucleic acid populations, wherein the nucleic acid molecules in each population having the same barcode sequence attached in proximity to the first end.   
     
     
         5 . The method of  claim 4 , wherein the attaching in (iii) is performed by PCR. 
     
     
         6 . The method of  claim 4 , wherein the attaching in (iii) is performed by ligation. 
     
     
         7 . The method of  claim 1 :
 wherein the first adapter comprises an outer polymerase chain reaction (PCR) primer region or nucleic acid amplification region and an inner sequencing primer region, and the barcode sequence.   
     
     
         8 . The method of  claim 4 , further comprising fragmenting a nucleic acid molecule comprising a target nucleotide sequence into a plurality of linear nucleic acid molecules prior to attaching the first adapter. 
     
     
         9 . The method of  claim 4 , wherein the nucleic acid molecules comprise first adapters attached at the 5′ and the 3′ ends, and wherein the first adapter attached at the 5′ end comprises a different barcode sequence than the first adapter attached at the 3′ end. 
     
     
         10 . The method of  claim 4 , wherein the nucleic acid molecules comprise first adapters attached at the 5′ and the 3′ ends, and wherein the first adapter attached at the 5′ end and the first adapter attached at the 3′ end comprise the same barcode sequence. 
     
     
         11 . The method of  claim 4 , wherein amplifying the plurality of target molecules comprises PCR. 
     
     
         12 . The method of  claim 11 , wherein amplifying the plurality of nucleic acid molecules is carried out using a primer complementary to the PCR primer region. 
     
     
         13 . The method of  claim 11 , further comprising removing the PCR primer region from the plurality of nucleic acid molecules after amplification. 
     
     
         14 . The method of  claim 13 , wherein the removing the PCR primer region is carried out before joining the first end with the second end to provide a plurality of circularized nucleic acid molecules. 
     
     
         15 . The method of  claim 1 , wherein fragmenting the plurality of clonal nucleic acid molecules at (b) is carried out by enzymatic, chemical or physical shearing. 
     
     
         16 . The method of any one of  claim 1 , wherein fragmenting the plurality of clonal nucleic acid molecules at (b) is carried out at random locations on the nucleic acid sequences. 
     
     
         17 . The method of  claim 83 , wherein the second adapter comprises two nucleic acid strands of different lengths, wherein the strand attached at the 5′ end of a fragmented nucleic acid molecule is of a different length than the strand attached at the 3′ end of the fragmented nucleic acid molecule, wherein one end of the second adapter is double stranded to facilitate ligation and the other end of the second adapter comprises a 3′ single-stranded overhang, and wherein only the longer of the two oligonucleotides comprises a sequence complementary to a second sequencing primer and comprises sufficient length to allow annealing of that primer. 
     
     
         18 . The method of  claim 83 , wherein replicating the nucleic acid molecules comprising the first and second adapters is carried out using two primers, the first of which is complementary to a constant sequence from the first adapter, and the second of which is complementary to an overhanging sequence of the second adapter, wherein the second adapter is an asymmetric adaptor, and wherein both adapters together add sequences necessary for nucleic acid sequencing. 
     
     
         19 . The method of  claim 18 , wherein the replicating is carried out using PCR. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 2 , wherein sequences from the plurality of clonal nucleic acid populations are sorted into independent groups is based on shared barcodes. 
     
     
         22 . The method of  claim 21 , wherein assembling each group is carried out independent of all other groups. 
     
     
         23 . The method of  claim 4 , further comprising selecting the plurality of nucleic acid molecules on the basis of size prior to attaching the first adapter. 
     
     
         24 . The method of  claim 1 , further comprising selecting the nucleic acid molecules on the basis of size prior to sequencing. 
     
     
         25 . The method of  claim 1 , wherein fragmenting the plurality of circularized nucleic acid molecules at step (d) comprises enzymatic, chemical or physical shearing. 
     
     
         26 . The method of  claim 13 , wherein the PCR primer region is removed by an enzyme that excises uracils and breaks the phosphate backbone. 
     
     
         27 . The method of  claim 13 , wherein the PCR primer region comprises methylated nucleotides and the PCR primer region is removed by restriction enzymes specific for methylated sequences. 
     
     
         28 . The method of  claim 1 , wherein nucleic acid sequence information is obtained for a longer nucleic acid sequence comprising a length of at least about 500 bases or at least about 1000 bases. 
     
     
         29 - 30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein nucleic acid sequence information is obtained for a longer nucleic acid sequence comprising a length from about 1 kilobase to about 20 kilobases. 
     
     
         32 . (canceled) 
     
     
         33 . The method of  claim 1 , wherein the nucleic acid sequence information comprises greater than about 95% fidelity to the target nucleotide sequence. 
     
     
         34 . The method of  claim 1 , wherein the target nucleotide sequence originates from genomic DNA. 
     
     
         35 - 82 . (canceled) 
     
     
         83 . The method of  claim 1 , further comprising after step (d) and before step (e):
 (i) attaching a second adapter to both ends of the plurality of fragmented nucleic acid molecules; and   (ii) replicating all or part of the plurality of nucleic acid molecules.   
     
     
         84 . The method of  claim 15 , wherein the physical shearing comprises acoustic shearing, sonication, or hydrodynamic shear. 
     
     
         85 . The method of  claim 15 , wherein the enzymatic shearing comprises a fragmentase, KAPA Frag Enzyme, DNase I, a non-specific nuclease, a transposase, a restriction endonuclease, or a tagmentase. 
     
     
         86 . The method of  claim 15 , wherein the enzymatic shearing comprises a double-stranded DNA fragmentase. 
     
     
         87 . The method of  claim 25 , wherein the physical shearing comprises acoustic shearing, sonication, or hydrodynamic shear. 
     
     
         88 . The method of  claim 25 , wherein the enzymatic shearing comprises a fragmentase, KAPA Frag Enzyme, DNase I, a non-specific nuclease, a transposase, a restriction endonuclease, or a tagmentase.