Phased Genome Sequencing

Abstract

The present disclosure provides methods for determining phased nucleic acid sequence for a single chromosome of interest and/or a single chromosomal fragment of interest. The present disclosure also provides methods for determining phased nucleic acid sequence for a plurality of single chromosomes of interest and/or a plurality of single chromosomal fragments of interest. The plurality of single chromosomes of interest may be of one chromosome type or of two or more chromosome types. The present disclosure also provides a method for isolating a plurality of chromosomal fragments of a specified size range, where the chromosomal fragments are from one or more specified regions of the genome. The plurality of chromosomal fragments may be separated into single chromosomal fragments and sequenced to provide phased nucleic acid sequence for the single chromosomal fragments. Alternatively, the plurality of chromosomal fragments may be sequenced together to provide unphased nucleic acid sequence for the chromosomal fragments.

1 - 15 . (canceled) 
     
     
         16 . A method for determining a phased nucleic acid sequence at a genetic locus, comprising:
 providing first and second fragments of first and second chromosomes of a chromosomal pair, respectively, the first and second fragments comprising the genetic locus;   separating the first and second fragments;   tagging polynucleotide products of an amplification of each of the chromosomal fragments, the polynucleotide products from each of the first and second chromosomal fragments comprising a different nucleic acid tag;   sequencing the polynucleotide products and associated tags; and   determining a phased nucleic acid sequence at the genetic locus in each of the first and second chromosomal fragments by attributing a sequence of the polynucleotide fragments to the first or second chromosomal fragment based upon the associated tag.   
     
     
         17 . The method of  claim 16 , wherein the fragments are separated into droplets. 
     
     
         18 . The method of  claim 16 , wherein the fragments are separated using fluorescence activated cell sorting (FACS). 
     
     
         19 . The method of  claim 16 , wherein the fragments are greater than 10 kb in length. 
     
     
         20 . The method of  claim 19 , wherein the fragments are between 1 and 50 Mb in length. 
     
     
         21 . The method of  claim 20 , wherein the fragments are between 1 and 40 Mb in length. 
     
     
         22 . The method of  claim 21 , wherein the fragments are between 1 and 20 Mb in length. 
     
     
         23 . The method of  claim 22 , wherein the fragments are between 1 and 10 Mb in length. 
     
     
         24 . The method of  claim 23 , wherein the fragments are between 1 and 5 Mb in length. 
     
     
         25 . A composition, comprising:
 a first container containing a reaction mixture, comprising:
 a first fragment of a first chromosome of a chromosomal pair; 
 reagents for carrying out an amplification reaction to produce first polynucleotide products, and for attaching first nucleic acid tags to the first polynucleotide products; and 
   a second container containing a reaction mixture comprising:
 a second fragment of a second chromosome of the chromosomal pair; and 
 reagents for carrying out an amplification reaction to produce second polynucleotide products, and for attaching second nucleic acid tags to the second polynucleotide products, the second nucleic acid tags being different from the first nucleic acid tags. 
   
     
     
         26 . The composition of  claim 25 , wherein each of the first and second fragments is greater than 10 kb in length. 
     
     
         27 . The composition of  claim 26 , wherein the first and second fragments are between about 1 and 50 Mb. 
     
     
         28 . The composition of  claim 27 , wherein the fragments are between 1 and 40 Mb in length. 
     
     
         29 . The composition of  claim 28 , wherein the fragments are between 1 and 20 Mb in length. 
     
     
         30 . The composition of  claim 29 , wherein the fragments are between 1 and 10 Mb in length. 
     
     
         31 . The composition of  claim 30 , wherein the fragments are between 1 and 5 Mb in length. 
     
     
         32 . The composition of  claim 25 , wherein the reagents for attaching the first and second tags to the first and second polynucleotide products comprise ligation reagents. 
     
     
         33 . A composition, comprising:
 a first plurality of polynucleotide products of an amplification of a first fragment of a first chromosome of a chromosome pair each of the first plurality of polynucleotide products comprising a first nucleic acid tag; and   a second plurality of polynucleotide products of an amplification of a second fragment of a second chromosome of a chromosome pair each of the second plurality of polynucleotide products comprising a second nucleic acid tag different from the first nucleic acid tag.