Paired End Sequencing

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Contents

Paired End Sequencing

The paired-end module of the GAII system allows both ends of a DNA fragment to be sequenced. This gives more specificity to alignment algorithms, especially in highly repetitive regions. The paired-end module allows for up to 36 bases of each end to be sequenced during the same sequencing run. Longer runs (up to 72nt) should be available in early 2009. http://illumina.com/downloads/ch2-ILMN_ProdGuide_DNAsequencing.pdf

Sample Submission Guidelines

Users are required to prepare their own libraries for sequencing by following the corresponding Illumina Protocols. If this is a service that you would like the BMC to perform in the future please visit the Ideas page.

Samples which are ready for sequencing should be gel-purified according to your protocol, and provided as at least 5uL of solution at a concentration of 10 nM in EB/ Tween 0.1%. To calculate your sample’s molar concentration, you must know the average length of your product.

When submitting a sample you must supply the BMC with the molar concentration, average length, and species of origin of all samples.

We encourage all those submitting samples to allow us to run QC analysis, using the 2100 BioAnalyzer, to obtain/confirm concentration information. Cyber green and qPCR QC analysis also coming soon!

Samples generated using standard kits with Illumina PCR primers can be submitted without sequencing primers. Custom sample preparations using customized PCR primer sequences should be provided along with a complementary sequencing primer.

Sample Submission Forms

Data

Images acquired from the solexa sequencer are processed through the bundled solexa inage extraction pipeline to get the sequence and quality score for each base. The data is aligned to a reference genome, if requested, using an interative ELAND algorithm. An in-depth QC report is included in the package. Sample QC report is here.

Turnaround Time

Each Genome Analyzer processes 8 samples per run, or 7 samples plus a control. The control is typically used to improve sequence quality. Full flowcells can be run usually within two weeks of submission. Partial submissions of less than eight samples (or 7 with control) are put into a project queue, where they join existing samples or await others before processing. Wait times for partial submissions vary depending on demand from other users.

Once the run has begun approximately 10 days are required for clustering, sequencing, and data analysis for a Paired End read (36bp read x 2).

Pricing

Illumina sequencing is currently available only for labs associated with the BioMicro Center Core departments. Sequencing is currently available at an introductory price:

      Paired End Reads (priced per lane):
           7 lane run | 8 lane run
                $1105 | $980 

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