Genomic Sequencing

• US 20090298064A1
• Filed: 05/29/2008
• Published: 12/03/2009
• Est. Priority Date: 05/29/2008
• Status: Abandoned Application

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Batzoglou, Serafim, Sundquist, Andreas, Ronaghi, Mostafa
• Application Number:
  US12/129,330
• Publication Number:
  US 20090298064A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Genomic sequencing is implemented for high throughput applications that can include short reads. In one example, whole-genome sequencing involves a method in which a subset of fragments of a target genome are selected as a random function, and each fragment is replicated into clones. The clones are ordered into clone contigs based on sets of overlapping clones, and potential read overlaps are determined from clone read data. The method can also involve reading local assemblies of contigs from regions smaller than a clone length and assembling the local assemblies into read sets, combining the assembled read sets into clone-sized regions and assembling the clone-sized regions, and assembling the clone-sized regions into clone contigs. Overlapping sets of clones and their ordering can be determined computationally from read data, with a high depth of clone coverage to provide a large number of boundaries on which the assemblies can be segmented into overlapping regions of pooled reads.

Claims

1. A method for genome sequencing, the method comprising:

as a random function, selecting a subset of fragments of a target genome;

replicating each fragment into clones;

ordering the clones into clone contigs based on sets of overlapping clones;

determining potential read overlaps from clone read data and validating base pairs of each read;

reading local assemblies of contigs from regions smaller than a clone length and assembling the local assemblies into read sets;

combining the assembled read sets into clone-sized regions; and

assembling the clone-sized regions into clone contigs.

2. The method of claim 1, further including tagging the cloned fragments with clone IDs, and using the clone IDs to identify a clone from which the read sets originate.

3. The method of claim 1, further including the step of identifying a clone from which the read sets originate based on uniquely tagged cloned fragments.

4. The method of claim 1, wherein reading local assemblies of contigs from regions smaller than a clone length includes:

finding all reads that overlap each particular clone,

performing intersection and subtraction operations on the sets of reads to isolate smaller regions, and

independently assembling each read set.

5. The method of claim 1, wherein selecting a subset of fragments of a target genome includes selecting a subset of fragments that cover the genome at high redundancy of at least about 4.0x coverage.

6. The method of claim 1, wherein selecting a subset of fragments of a target genome includes selecting a subset of fragments that cover the genome at high redundancy of at least about 4.0x coverage, and further including

acquiring sequencing reads from fragments at redundancy that is lower than said high redundancy, and

constructing the read sets by combining sequencing reads acquired from different overlapping fragments and assembling into local assemblies.

7. The method of claim 1, wherein validating includes

comparing overlapping read data from the sequence to detect overlapping reads that are different for common data, and

performing error correction on the overlapping reads that are detected as being different.

8. The method of claim 1, wherein validating includes detecting overlapping reads that are different for common data and performing error correction on the overlapping reads that are detected as being different.

9. A method for genome sequencing that uses validated clones generated from a subset of fragments of a target genome and ordered into clone contigs based on sets of overlapping clones, the method comprising:

reading local assemblies of contigs from validated clone regions smaller than a clone length and assembling the local assemblies into read sets;

combining the assembled read sets into clone-sized regions; and

assembling the clone-sized regions into clone contigs.

10. The method of claim 9, further including the step of providing the validated clones by detecting overlapping reads that are different for common data and performing error correction on the overlapping reads that are detected as being different.

11. The method of claim 9, wherein the step of assembling the clone-sized regions into clone contigs includes assembling the clone-sized regions into an entire clone contig.

12. The method of claim 9, wherein the step of assembling the clone-sized regions into clone contigs includes assembling the clone-sized regions for a genome.

13. The method of claim 9, further including the step of providing the validated clones and wherein validating includes

comparing overlapping read data from the sequence to detect overlapping reads that are different for common data, and

performing error correction on the overlapping reads that are detected as being different.

14. The method of claim 9, wherein reading local assemblies of contigs from regions smaller than a clone length includes:

finding all reads that overlap each particular clone,

performing intersection and subtraction operations on the sets of reads to isolate smaller regions, and

independently assembling each read set.

15. The method of claim 14, wherein each read includes raw data read from the sequence

16. A storage device comprising data representing computer-executable instructions that, in response to being accessed and executed by a computer, cause performance of a method for genome sequencing that uses validated clones generated from a subset of fragments of a target genome and ordered into clone contigs based on sets of overlapping clones, the method including the steps of:

reading local assemblies of contigs from validated clone regions smaller than a clone length and assembling the local assemblies into read sets;

combining the assembled read sets into clone-sized regions; and

assembling the clone-sized regions into clone contigs.