HUMAN EXOME SEQUENCING

Targeted sequencing of human exons is an innovative technique to identify causative mutations for genetic disorders quickly in a cost-effective manner. BaseClear uses Roche Nimblegen SeqCap EZ Exome capture technology together with Illumina sequencing, allowing you to sequence the coding portion of the human genome for a fraction of the cost of whole-genome sequencing. 

Research has shown that the majority of disease-causing mutations occur in the protein-coding portion of the human genome, also called the exome. The human exome is therefore often referred to as the most relevant portion of the genome. The exome contains all coding exons and comprises about 1% of the human genome. For this reason, it is interesting to use targeted sequencing to sequence only this specific portion of the genome. This approach allows researchers to focus on these genomic regions of interest to discover causative mutations, providing a more efficient way to perform studies with numerous samples. This can be accomplished via the Sequence Capture method of Roche Nimblegen, in combination with Illumina HiSeq 2000 sequencing.

Sequence Capture protocol The Sequence Capture was developed to eliminate the necessity of setting up thousands of PCR reactions, instead allowing for parallel enrichment of target regions in one single experiment. In a first step, a sequence cap EZ oligo pool is made against target regions in the genome (see, figure, stage 1). A standard shot-gun library is made from genomic DNA (stage 2), and hybridized to the SeqCap EZ oligo pool (stage 3). Streptavidin beads are used to pull down the complexes of capture oligos and genomic DNA fragments (stage 4). Unbound fragments are removed by washing (stage 5). The enriched fragment pool is amplified by PCR (stage 6). Subsequently, the success of the enrichment is checked by qPCR at control loci (stage 7). Finally, the end product is a sequencing library enriched for target regions, which will be sequenced by high throughput sequencing. The combination of SeqCapEZ Exome with next generation sequencing results in high performance data. The enrichment results in a 30-fold reduction of sequencing, leading to a better coverage with less sequencing.

Certified Service Provider (CSPro)
From early in 2011, BaseClear will be a Certified Service Provider for Roche Nimblegen. This certification enables BaseClear to offer customers a complete set of DNA target enrichment services. Exome enrichment has become a very important technique over the past few years, particularly for human genome research.

Approach

Our product specialist will work with you to determine which sample preparation and run type is best to perform.

Starting material

• 20 ug genomic DNA (minimal concentration 200 ng/ul).

Enrichment and sequencing

• Enrichment with the Roche Nimblegen SeqCap EZ Exome Library
• Sequencing with the Illumina HiSeq 2000

Output and further bioinformatics options

• You will receive guaranteed 3 Gb of DNA sequence data per exome.
• The raw sequencing data will be provided in either fastq format.
• Are you interested in further downstream analysis such as SNP detection or reference alignment? If so, please take a look at BaseClear’s additional NGS analysis services or contact our Bioinformatics Product specialists for further details.
• The bioinformatics strategy will be determined in close collaboration between the customer and the bioinformatics and Genome Analysis product specialists.