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Methods and compositions for large-scale analysis of nucleic acids using dna deletions

This application claims priority to provisional application Ser. No. 60/864,992, filed Nov. 9, 2006, which is hereby incorporated by reference in its entirety.

Large-scale sequence analysis of genomic DNA is central to understanding a wide range of biological phenomena related to states of health and disease both in humans and in many economically important plants and animals, e.g., Collins et al (2003), Nature, 422: 835-847; Service, Science, 311: 1544-1546 (2006); Hirschhorn et al (2005), Nature Reviews Genetics, 6: 95-108; National Cancer Institute, Report of Working Group on Biomedical Technology, “Recommendation for a Human Cancer Genome Project,” (February, 2005); Tringe et al (2005), Nature Reviews Genetics, 6: 805-814. The need for low-cost high-throughput sequencing and re-sequencing has led to the development of several new approaches that employ parallel analysis of many target DNA fragments simultaneously, e.g., Use of water/buffer-in-oil emulsions to carry out enzymatic reactions is well known in the art, particularly carrying out PCRs, e.g., as disclosed by Drmanac et al., Scienta Yugoslavica, 16(1-2): 97-107 (1990), Margulies et al, Nature, 437: 376-380 (2005); Margulies et al, Nature, 437: 376-380 (2005); Shendure et al (2005), Science, 309: 1728-1732; Metzker (2005), Genome Research, 15: 1767-1776; Shendure et al (2004), Nature Reviews Genetics, 5: 335-344; Lapidus et al, U.S. patent publication US 2006/0024711; Drmanac et al, U.S. patent publication US 2005/0191656; Brenner et al, Nature Biotechnology, 18: 630-634 (2000); and the like.

Such approaches reflect a variety of solutions for increasing target polynucleotide density in planar arrays and for obtaining increasing amounts of sequence information from each application of a sequence detection reaction.

Most traditional methods of sequence analysis are restricted to determining a few tens of nucleotides before signals become significantly degraded, thus placing a significant limit on overall sequencing efficiency. Such short sequence reads are particularly problematic in regions of a target sequence which contain long strings of repeating nucleotides or tandem repeats.

In view of such limitations, it would be advantageous for the field if methods and tools could be designed to increase the efficiency of sequencing reactions as well as the efficiency of assembling complete sequences from shorter read lengths.

In one aspect, the invention provides a method for forming a polynucleotide that includes a deletion mate pair. This method includes the step of providing a first linear construct, which includes a first adaptor interposed between a first target polynucleotide fragment and a second target polynucleotide fragment. The first target polynucleotide fragment and the second target polynucleotide fragment are contiguous nucleic acids within a target polynucleotide. In a further step, a deletion adaptor is ligated to the first linear construct to form a second linear construct. This deletion adaptor includes a recognition site for a restriction endonuclease, and the restriction endonuclease in the deletion adaptor cleaves at a known distance from its recognition site. The restriction endonuclease is applied to cleave the second linear construct to form a third linear construct, thus forming the polynucleotide that includes a deletion mate pair.

In another aspect, the invention provides a method for forming a circular polynucleotide that includes a deletion mate pair. This method includes the step of providing a first circular construct. The first circular construct includes a first adaptor and a target polynucleotide. The first adaptor includes a recognition site for a first restriction endonuclease that cleaves at a known distance from the recognition site and a recognition site for a second restriction endonuclease that cleaves within the first adaptor. The first restriction endonuclease is used to cleave the first circular construct to form a first linear construct. The first linear construct is in turn cleaved with the second restriction endonuclease to form a second linear construct. The second linear construct is then circularized to create a second circular construct, thus forming the circular polynucleotide that includes a deletion mate pair.

In yet another aspect, the invention provides a method for forming a polynucleotide that includes a deletion mate pair. This method includes the step of providing a first linear construct. This first linear construct includes a target polynucleotide and an adaptor, and in addition, a first adaptor is attached to one end of the polynucleotide. A deletion adaptor is ligated to the end of the first linear construct opposite the first adaptor, and the deletion adaptor includes a recognition site for a restriction endonuclease that cleaves at a known distance from the recognition site. The restriction endonuclease is applied to cleave the first the first linear construct to form a second linear construct, thus forming the polynucleotide that includes a deletion mate pair.

In still another aspect, the invention provides a method for forming a polynucleotide that includes a deletion mate pair. This method includes the step of providing a first linear construct which includes a target polynucleotide. A deletion adaptor is ligated to one end of the first linear construct, and the deletion adaptor includes a recognition site for a restriction endonuclease that cleaves at a known distance from the recognition site. The first linear construct is cleaved with the restriction endonuclease to form a second linear construct, thus forming the polynucleotide that includes a deletion mate pair.

In another aspect, the invention provides a method for forming a polynucleotide that includes a deletion mate pair. This method includes the step of providing a first linear construct that includes a target polynucleotide. A deletion adaptor is ligated to one end of the linear construct, and this deletion adaptor comprises a recognition site for a restriction endonuclease that cleaves at a known distance from the recognition site. The first linear construct is cleaved the restriction endonuclease to form a second linear construct, thus forming the polynucleotide that includes a deletion mate pair.

In still another aspect, the invention provides a method for forming a polynucleotide that includes a deletion mate pair. The method includes the step of providing a first circular construct. The first circular construct includes a first adaptor and a target polynucleotide, and the first adaptor includes a recognition site for a first restriction endonuclease that cleaves at a known distance from the recognition site. The first circular construct is then cleaved with the first restriction endonuclease to form a first linear construct. In a further step, a second adaptor is provided, and the second adaptor includes a recognition site for a second restriction endonuclease that cleaves at a known distance from the recognition site. The second adaptor is ligated to one end of the first linear construct to create a second linear construct, the second linear construct is then circularized to form a second circular construct, thus forming the polynucleotide that includes a deletion mate pair.

In one aspect of the invention, precise mate pair deletion constructs comprise a deletion of a specific length (e.g., about 10-100 or more bases) or a series of deletions of known length multiples, e.g., a set of constructs comprising constructs with a known 10 nt deletion constructs with a known 20 nt deletion, constructs with a known 30 nt deletion. Such precise mate pair deletion constructs can be used to extend read lengths, by cleaving circularized target nucleotides, deleting a known number of bases at the cleavage site, identifying bases on each side of the deletion, and analyzing the combined data of the precise mate pair constructs to form an indirectly extended read length comprising of both directly determined and deleted bases.

In another aspect of the invention, sequencing reactions using precise deletion mate pair constructs and conventional mate pair constructs are utilized. Preferably, the sequencing reads of the combined nucleotides will span the length of the known deletion in any of the deletion mate pair constructs.

In one aspect of the invention, a library of constructs are prepared, wherein the library comprises staggered restriction fragments, with each fragment comprising a defined deletion on one or both sides of the fragment. Sequencing reads from these libraries provide longer combined read lengths than the use of the fragments alone. These library constructs may comprise both precise deletion mate pairs and/or traditional mate pairs.

In one aspect, the invention provides a method for analyzing a polynucleotide sequence. This method includes providing a deletion mate pair construct. In a preferred aspect, the deletion mate pair construct includes the following: (i) a first adaptor, (ii) a second adaptor, (iii) a first target sequence, and (iv) a second target sequence. The first target sequence and the second target sequence span a portion of the polynucleotide sequence. The method includes the step of identifying at least one nucleotide of the first target sequence and at least one nucleotide of the second target sequence, thereby analyzing the polynucleotide sequence.

In one aspect, the invention provides a method for forming a library of a plurality of circularized deletion mate pair constructs. This method includes ligating a deletion adaptor to each of a plurality of first linear constructs. The deletion adaptor includes a recognition site for a restriction endonuclease that cleaves at a known distance from the recognition site. At least a portion of the plurality of first linear constructs is cleaved with the restriction endonuclease to provide a plurality of second linear constructs. At least a portion of the plurality of the second linear constructs is circularized, thus forming the library of circularized deletion mate pair constructs.

In one aspect, the invention provides a method for forming a random array. In this method, a support with a surface is provided, as is a plurality of deletion mate pair constructs. The plurality of deletion mate pair constructs is immobilized on the surface, thereby forming the random array. In a further aspect the invention provides random arrays made according to this method.

In another aspect, the invention provides a library that includes a plurality of deletion mate pair constructs. The plurality of deletion mate pair constructs include target sequences, and the target sequences together represent at least about 80% of a genome.