Nucleotide sequence from amplicon subfragments
The invention provides methods and kits for ordering sequence information derived from one or more target polynucleotides. In one aspect, one or more tiers or levels of fragmentation and aliquoting are generated, after which sequence information is obtained from fragments in a final level or tier. Each fragment in such final tier is from a particular aliquot, which, in turn, is from a particular aliquot of a prior tier, and so on. For every fragment of an aliquot in the final tier, the aliquots from which it was derived at every prior tier is known, or can be discerned. Thus, identical sequences from overlapping fragments from different aliquots can be distinguished and grouped as being derived from the same or different fragments from prior tiers. When the fragments in the final tier are sequenced, overlapping sequence regions of fragments in different aliquots are used to register the fragments so that non-overlapping regions are ordered. In one aspect, this process is carried out in a hierarchical fashion until the one or more target polynucleotides are characterized, e.g. by their nucleic acid sequences, or by an ordering of sequence segments, or by an ordering of single nucleotide polymorphisms (SNPs), or the like.
1. A method of characterizing nucleotide sequences of one or more target polynucleotides, the method comprising the steps of:
providing a plurality of aliquots, at least some of which comprise one or more fragments of the target polynucleotide(s);
amplifying the fragments in the aliquots to produce first amplicons;
randomly fragmenting the amplicons in the aliquots to produce subfragments;
incorporating oliqonucleotide tags of known sequence into at least some of the subfragments in at least some of the aliquots, whereby the tag identifies the aliquot;
obtaining sequence reads of the target polynucleotides and the oliqonucleotide tag of at least some of the subfragments; and
assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s), wherein the assembling comprises grouping the sequence reads according to the sequence of the oliqonucleotide tags.
2. The method of claim 1 , wherein any given portion of the target polynucleotide(s) is represented by a single non-overlapping fragment in sixty percent or more of the aliquots containing such portion.
3. The method of claim 1 , wherein at least sixty percent of the aliquots containing more than one fragment contain only non-overlapping fragments.
4. The method of claim 1 , wherein at least ninety percent of the aliquots containing more than one fragment contain only non-overlapping fragments.
5. The method of claim 1 , comprising pooling tagged subfragments from a plurality of the aliquots before obtaining the sequence reads.
6. The method of claim 1 , wherein the step of obtaining nucleic acid sequence reads comprises:
amplifying the subfragments to produce second amplicons;
forming a regular array in which the second amplicons are fixed to a surface; and
obtaining sequence reads from the second amplicons on the array.
7. The method of claim 6 , wherein the second amplicons are fixed to the surface in a rectilinear or hexagonal pattern.
8. The method of claim 1 , wherein providing the plurality of aliquots comprises fragmenting and amplifying genomic DNA from a sample, and then separating the fragmented and amplified genomic DNA into the aliquots.