Methods for single-molecule analysis

Abstract

Methods for single-molecule preparation and analysis are disclosed herein. The methods can, for example, be used for isolating and analyzing DNA from various biological samples.

Description

BACKGROUND[0001]1. Technical Field[0002]The present invention relates to the field of nanotechnology and to the field of single molecule genomic analysis.[0003]2. Description of the Related Art[0004]Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data. However, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable.[0005]Accurate de novo assembly of sequence reads represents the weak link in genome projects despite advances in high-throughput sequencing [1,2]. There are two general steps in genome sequence assembly, generation of sequence contigs and scaffolds, and their anchorin...

AI Technical Summary

Benefits of technology

This patent is about methods for preparing samples and performing single molecule analysis. The technical effects include methods to improve information density for genome mapping and mitigate the effects of fragile sites.

Problems solved by technology

However, de novo genome assembly remains a great challenge, particularly for large genomes.

NGS short reads are often insufficient to create large contigs that span repeat sequences and facilitate unambiguous assembly.

Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable.

NGS reads are often too short for unambiguous assembly.

Paired-end reads can bridge contigs into scaffolds, but there are often gaps within the scaffolds.

Contigs and scaffolds may be difficult to map if they are too short compared to the map resolution.

Additionally, there are errors in the contigs and scaffolds themselves, often due to misassembly of repeat sequences.

Typical medium to large genomes contain 40-85% repetitive sequences [5-8], dramatically hindering effective de novo sequence assembly.

However, information density for optical maps is only about one site per 20 kb, and the technology is limited in utility by high error-rates, non-uniform DNA linearization, and low throughput.

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