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De novo Sequencing

De novo sequencing is used to sequence uncharacterized genomes where there is no reference sequence available, or known genomes where significant structural variation is expected. De novo sequence reads are typically generated on the Roche* 454 GS FLX Titanium (taking advantage of the long read lengths) or Illumina* HiSeq 2000 platforms. Sequence reads from multiple platforms can also be combined (e.g., Illumina + 454) for cost effective coverage of large genomes, or with Sanger sequencing reads for genomes with particularly abundant or complex repeats.

Applications

• Sequencing of unkown genomes or when no reference sequence is available
• Samples with expected large structural variation e.g. cancer cells
• Microbial sequencing – experimental strains, genomes with high plasticity

Workflow

Samples

Sequencing can be carried out from a variety of different starting materials including:

• Bacterial, viral, phage, fungus, eukaryote genomic DNA
• BACs, fosmids
• Fragmented DNA

Library Construction

Library construction is carried out to prepare the DNA for sequencing, including size selection and barcoding of fragments as required. The type of library needed will be project dependent, taking into consideration the application and genome characteristics to ensure optimal coverage and assembly.

Library options:

• Fragment and paired end libraries – for cost-effective, deep coverage
• Mate pair libraries – for scaffolding and detection of structural variants

Sequencing

• Roche 454 GS FLX Titanium – for whole genomes and fragmented DNA
• Illumina HiSeq 2000 – for whole genomes and fragmented DNA
• Roche 454 GS FLX Titanium and Illumina HiSeq 2000 – for repetitive genomes
• Roche 454 GS FLX Titanium and ABI 3730XL – for large or complex genomes

Finishing/Gap Closure

If finishing is required, sequence gaps and areas of low sequence quality will be finished with PCR^† amplification and primer walking. To ensure complete genome coverage, and for efficient gap closure, we can also perform scaffold and physical gap closure with Sanger sequencing, using custom primer walking and direct paired end sequencing for repeat and difficult sequences.

Data Analysis

A variety of data analysis services are available depending on your requirements. Custom reports can be generated as well as teleconferences setup with our scientists to talk you through the findings.

Data analysis options include:

• Sequence assembly
• Scaffold assembly and orientation
• In Silico finishing
• Annotation: ORF detection, CDS detection, automatic annotation by transfer of existing annotated closely related genome

Results are delivered via a secure FTP site or are shipped on a portable hard drive depending upon the size of the dataset.

Getting Started

Beckman Coulter Genomics project managers will work with you to ensure appropriate study design for your project. This design will accommodate your specific needs and include recommendations for sample preparation and data analysis options. Please contact Beckman Coulter Genomics to discuss your next project.

Publications: De novo sequencing

Genome Sequence of Taylorella equigenitalis MCE9, the Causative Agent of Contagious Equine Metritis
J. Bacteriol., April 2011, p. 1785, Vol. 193, No. 7

Complete Genome Sequence of a Free-Living Vibrio furnissii sp. nov. Strain (NCTC 11218)
J. Bacteriol., Mar 2011; 193: 1487 - 1488.

Frameshift Mutations in a Single Novel Virulence Factor Alter the In Vivo Pathogenicity of Chlamydia trachomatis for the Female Murine Genital Tract
Infect. Immun. Sep 2010; 78: 3660 - 3668.

Marine Prasinovirus Genomes Show Low Evolutionary Divergence and Acquisition of Protein Metabolism Genes by Horizontal Gene Transfer
Journal of Virology, December 2010, p. 12555-12563, Vol. 84, No. 24

Microbisporicin gene cluster reveals unusual features of lantibiotic biosynthesis in actinomycetes
PNAS. July 27, 2010 vol. 107

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