De novo sequencing of an unknown organism

Customized strategies to specifically answer your scientific question

Your genome project

Determination of the genome sequence of an unknown organism to facilitate biological or medical research like e.g. revealing novel sequences and genes and characterizing of functional potential.
 

Strategy

Depending on the aim of the project (required level of finishing), expected size and complexity of the genome different sequencing strategies can be applied. From backbone sequencing using one technology to multiplatform approaches combining the newest Next Gen sequencing technologies. Obtain high quality results through sequencing of different paired end and mate pair libraries and following state-of-the-art bioinformatics.
 

Starting material

• Genomic DNA
• BAC or fosmid libraries if available

Small genomes

The high raw read accuracy and the possibility to combine different paired end and mate pair insert sizes make the HiSeq 2000 a suitable technology for the cost-effective de novo sequencing of small genomes like e.g., bacteria, viruses and small fungi (up to 37 Mb). Through combination with optimized short read assembly algorithms and tools successful de novo assemblies can be performed.
 

Sequencing

• Illumina HiSeq 2000 I paired end reads for backbone sequencing (80x coverage)

• Scaffolding & contig orientation through sequencing of large insert mate pair libraries

• Finishing or gap closure through PCR on plasmid/fosmid clones followed by Sanger sequencing on ABI 3730xl

Bioinformatics

• Raw data (fastq)

• Sequence of assembled (and scaffolded) contigs (fasta)

• Assembly report (txt)

• Scaffolding metrics (txt)

Large genomes

Long sequencing reads are needed to resolve repetitive sequences in large and complex genomes and to hence enable a high quality assembly. Large insert mate pair libraries allow to further improve the assembly as they enable the ordering and orientation of the resulting contigs.

Sequencing

• Roche GS FLX+ I single reads for backbone sequencing (10 to 15x coverage)

• Scaffolding & contig orientation through sequencing of large insert mate pair libraries

• Improvement of sequence quality and correction of homopolymers through additional Illumina HiSeq 2000 data

• Finishing or gap closure through PCR on plasmid/fosmid clones followed by Sanger sequencing on ABI 3730xl

Bioinformatics

• Raw data (fasta and fastq)

• Flowgrams (sff)

• Semiautomatic GS FLX+ Assembly (ace)

• Sequence of assembled (and scaffolded) contigs (fasta)

• Assembly report (txt)

• Scaffolding report (txt)

• Assembled contigs coverage report (txt)

• Annotation

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