Next Generation Sequencing

We use the Roche/454 GS-FLX Titanium system to accomplish a variety of experimental goals. These include whole genome de novo sequencing, characterization of transcriptomes, analyses of genome variation (polymorphism detection, population studies), metagenomics, and mapping of protein-nucleic acid interactions, sites of epigenetic modification, and chromatin interaction.

Process
For each application, a customized sequencing library is prepared. Input samples include whole genomic DNA, RNA, fractionated genomic samples (from experiments such as ChIP, bisulfite treatment, etc.), PCR products and other DNA or RNA fragments. Library templates to be sequenced are immobilized individually onto beads and clonally amplified in water-in-oil emulsion microreactors. Nucleotide additions are recorded by high-resolution imaging of pyrosequencing reactions on a dense plate of bead-bound templates. Data are provided as sequenced reads assembled into contigs. If a paired-end library is sequenced, contigs can be oriented as scaffolds in the assembly

Throughput
Currently, nearly 1 million high-quality reads can be sequenced simultaneously, yielding ~400-600 Mb of data in a single run. Experiments requiring less sequence coverage can be run cost-effectively on a subdivided plate. For some samples, a unique sequence identifier ("bar code") can be incorporated during library preparation, allowing multiple libraries to be mixed together in one run. With a 400-nucleotide per read average, the Roche/454 GS-FLX Titanium system provides accurate reads more than 10 times longer than those generated from other high-throughput sequencing technologies, making it the platform of choice for the assembly and study of uncharacterized genomes.

CGB Sequencing Unit

The Center for Genomics and Bioinformatics offers proven expertise for all aspects of high-throughput sequencing projects. We are a research group with experience in customizing experiments and developing new applications. By optimizing library synthesis and instrument use, we provide to researchers data that routinely exceed quality expectations of GS-FLX output. With extensive experience in data analysis, we truly complete the experiment with computational support.

If you are considering high-throughput sequencing for your research, please contact us for consultation on experimental design, pricing and scheduling: 454 at cgb.indiana.edu

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