Methods for single-molecule analysis

Inactive Publication Date: 2014-08-07
BIONANO GENOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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.

Method used

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  • Methods for single-molecule analysis
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  • Methods for single-molecule analysis

Examples

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example 1

[0054]E. coli genomic DNA was nicked with Nt.BspQI nicking endonuclease. The nicked DNA was labeled with Taq polymerase by nick translation using Atto dUTP or Alexa dUTP in the presence of cold dATP, dGTP, and dCTP. The labeled nicks were: 1.) not repaired, 2.) repaired with PreCR as recommended by manufacture (New England BioLabs), 3.) repaired with PreCR under conditions of omitting dGTP, 4.) repaired with PreCR under conditions of omitting dATP and dGTP, or 5.) repaired with Taq polymerase under conditions of omitting dGTP. Ligation was then performed with a ligase. The resulting DNA was stained with YOYO-1 (Life Technologies) and processed on the Irys system (BioNano Genomics). Briefly, DNA was linearized in massively parallel nanochannels, excited with the appropriate laser for backbone and label detection, and optically imaged. Mapping to a reference genome, center of mass, and False Positive (FP) and False Negative (FN) calculations were carried out using nano Studio data ana...

example 2

[0055]E. coli genomic DNA was nicked with Nt.BspQI nicking endonuclease. The nicked DNA was labeled with Taq polymerase by nick translation using Atto dUTP. The labeled DNA was: 1.) left unrepaired or 2.) treated with FEN Ito remove flaps followed by a ligase to repair the translated nicks. The DNA was linearized in massively parallel nanochannels, excited with the appropriate laser for backbone and label detection, and optically imaged. Mapping to a reference genome, center of mass, and False Positive (FP) and False Negative (FN) calculations were carried out using nano Studio data analysis software (BioNano Genomics). Results are shown in FIG. 3.

example 3

[0056]Drosophila genomic DNA was nicked with Nt.BspQI or Nb.BbVCI nicking endonuclease. The nicked DNA was labeled with Taq polymerase by nick translation using Atto dUTP. The labeled DNA was treated with PReCR reagent (New England Biolabs) to repair the nicks. The resulting DNA was stained with YOYO-1 (Life Technologies) and processed on the Irys system (BioNano Genomics). Mapping to a reference genome, center of mass, and False Positive (FP) and False Negative (FN) calculations were carried out using nano Studio data analysis software (BioNano Genomics). Results are shown in FIG. 4.

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6874C12Q1/6809C12Q2521/301C12Q2565/1025C12Q1/6806C12Q1/6869
Inventor CAO, HANXIAO, MINGHASTIE, ALEXSAGHBINI, MICHAEL G.SADOWSKI, HENRY B.
Owner BIONANO GENOMICS
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