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. Illumina Paired End sample preparation kits can be purchased directly from Illumina or through the BioMicro Center for $3,600.00 plus shipping costs (10 sample preps per kit). To request an order or for more information please contact Ali Perrotta
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 at least 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 to obtain/confirm concentration information. For more information on our QC methods please see the Sequencing Quality Control page.
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.
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.
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).
Illumina sequencing is currently available only for labs associated with the BioMicro Center Core departments.
|ILLUMINA SEQUENCING||CORE LAB/MIT||Non-MIT||unit||Notes|
|Paired End (36+36nt)||$1,450||$2,175||per lane||Includes quality control (RT-PCR and BioAnalyzer), sequencing, genome alignment and data storage of Firecrest files for 2 yrs.|
|Add'l 36nt||$300||$450||per lane|
|Sample Multiplexing||$50||$75||per 10 samples||Combined over the whole order. Only applies to samples combined by BCM technicians.|
|Add'l Repriming||$300||$450||per flow cell||Uses 6nt. Cost is divided by number of lanes requiring repriming. Repriming is required for standard Illumina multiplexing.|