BioMicroCenter:Expression Seq
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RNA Seq
We offer a mRNA Sequencing service utilizing the Genome Analyzer System from Illumina (Solexa). This data provides a much more detailed view of the steady-state transcriptome in a population of cells and provides more dynamic range then microarray hybridization.
Primarily, we offer:
- Digital Gene Expression Sequencing
Digital Gene Expression is similar to SAGE analysis. The sequencing of short mRNA “tags” provides genome-wide expression profiles and is used as an alternative to full-genome microarray hybridization or qPCR. Digital Gene Expression is more sensitive, has a greater dynamic range, and less background than traditional microarrays. It allows for more accurate quantification and detection of very low abundance transcripts and does not rely on prior sequence knowledge. http://illumina.com/downloads/rnaDGETagProfiling.pdf
- Whole Transcriptome Analysis
Sequencing the entire transcriptome can reveal valuable information about exon boundaries and splice variants that may be missed by microarray analysis or Digital Gene Expression. Kits for whole transcriptome analysis are expected to be available from Illumina soon.
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 mRNA-seq sample preparation kits can be purchased through the BioMicro Center for $2,000.00 plus shipping costs (8 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.
Data
Images acquired from the Solexa sequencer are processed through the bundled Solexa image extraction pipeline to get the sequence and quality score for each base. The data is aligned to a reference genome, if requested, using an interactive 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 six days are required for clustering, sequencing, and data analysis for a 36 base pair read. The process requires approximately 5 days for a 25 base pair read and 10 days 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.
| ILLUMINA SEQUENCING | CORE LAB | Non-MIT | unit | Notes |
|---|---|---|---|---|
| Single End (36nt) | $805 | $1,200 | per lane | Includes quality control (RT-PCR or BioAnalyzer, sequencing, genome alignment and data storage of Firecrest/IPAR files for 2 yrs. |
| Paired End (36+36nt) | $1,375 | $2,000 | per lane | |
| Add'l 36nt | $295 | $370 | per lane | |
| Illumina Multiplexing | $200 | $300 | per flow cell | Uses 6nt. Cost is divided by number of lanes requiring the multiplex reads. |
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RNA Seq Literature
<biblio>
- Paper1 pmid=18978772
- Paper2 pmid=19015660
- Paper3 pmid=18550803
