Methods and Compositions for Large-Scale Analysis of Nucleic Acids Using DNA Deletions

Abstract

The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are constructs that include pairs of target sequences which are separated by a known distance in the polynucleotide from which they are derived.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]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.BACKGROUND OF THE INVENTION[0002]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 simu...

AI Technical Summary

Benefits of technology

[0012]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.

[0013]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.

[0014]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.

Problems solved by technology

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.

Images