Sequencing by synthesis based ordered restriction mapping

• US 20070082358A1
• Filed: 10/10/2006
• Published: 04/12/2007
• Est. Priority Date: 10/11/2005
• Status: Abandoned Application

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Fuerst, Roderic, Kopal, Guido
• Application Number:
  US11/545,368
• Publication Number:
  US 20070082358A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6841 In situ hybridisation

Abstract

The present invention is directed to a method for de novo assembly of genomic sequence information comprising the combination optical whole genome restriction mapping and ultra high throughput pyrosequencing.

Claims

1. A method for de novo assembly of genomic sequence information comprising the steps of:

(i) providing genomic DNA isolated from a specific organism;

(ii) generating sequence information from the genomic DNA by

clonally isolating and amplifying the genomic DNA to produce a library of single stranded DNA molecules,

sequencing the library by a sequencing by synthesis reaction in order to create whole genome shotgun sequence information, and

assembling contigs from the sequence reads obtained from the whole genome shotgun sequence information;

(iii) obtaining whole genome optical restriction map information for the organism'"'"'s genomic DNA for at least one restriction enzyme and generating an ordered restriction map; and

(iv) aligning the sequence information obtained from step (ii) such that the sequence contigs are orientated and ordered with respect to the ordered restriction map obtained in step (iii).

2. The method of claim 1 wherein the genomic DNA is size fractionated, and fragments of a smaller size are used to generate sequence information according to step (ii), whereas fragments of a larger size are used to generate an ordered restriction map according to step (iii).

3. The method of claim 1 further comprising the steps of

identifying at least one sequence gap which is not covered by a contig, and

determining the length of the sequence gap.

4. The method of claim 3 further comprising the steps of

identifying appropriate primer sequences capable of amplifying a DNA fragment covering the sequence gap,

performing a PCR reaction with a mixture comprising the primers, a Taq DNA polymerase, and a thermostable DNA polymerase with proofreading activity to amplify the DNA fragment, and

sequencing the DNA fragment.

5. The method of claim 1 further comprising the step of validating the contigs obtained from step (ii) based on the ordered restriction map obtained in step (iii).

6. The method of claim 5 further comprising

identifying a contig which is not validated by the ordered restriction map obtained in step (iii) and

reassembling the sequence reads obtained in step (ii) without allowing recreation of the contig which has failed to pass the validity test.

8. The method of claim 1 wherein the step of clonally isolating and amplifying the genomic DNA comprises the steps of

randomly fragmenting the isolated genomic DNA,

ligating adaptors to the ends of the genomic DNA fragments,

capturing the adaptor modified genomic DNA fragments onto a bead via the adaptors,

mixing the genomic fragment bearing solid substrates together with amplification reagents and an oil to form an emulsion, and

conducting PCR amplification of the genomic DNA within each hydrophilc droplet, wherein the newly synthesized strands remain within their droplets and are bound to the respective bead.

7. A computer program product comprising a software program to compare and/or align a whole genome ordered restriction map with multiple contigs obtained from a sequencing by synthesis reaction.