BioMicroCenter:Small RNA Seq

Small RNA Seq
The BioMicroCenter supports small RNA sequencing (small RNA-seq) using the Illumina platform. Small RNA Seq can be helpful in identifying novel small RNA molecules and the relative quantification of microRNAs from different samples. Unlike more traditional small RNA characterization techniques, such as qPCR and microarray hybridization, the Illumina platform is not limited by a previous knowledge of sequence and can resolve closely related family members. small RNA seq libraries are typically run as 36nt single end reads. http://illumina.com/downloads/rnaDGESmallRNA_Datasheet.pdf

In interpreting small RNA-seq results it is important to note the results of a recent study, Linsen, 2009 PMID: 19564845. In this study it is stated, in disagreement with previous studies, that small RNA-seq can not accurately portray complete small RNA quantification due to inherent biases. Data obtained from small RNA-seq, microarray hybridization, qPCR and other Next Generation Sequencing techniques should therefore be interpreted as relative quantifications.

Sample Submission Guidelines
Users are required to prepare their own libraries for sequencing by following the corresponding Illumina Protocols. Illumina small RNA sample preparation kits can be purchased directly from Illumina or through the BioMicro Center. To request an order or for more information please contact Kevin Thai

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.

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 Illumina sequencer are processed through the bundled Illumina image extraction pipeline to get the sequence and quality score for each base. The data is aligned to known genomic loci's of all miRNA looking for an exact match. An in-depth QC report is included in the package.

Turnaround Time
Each Genome Analyzer processes 7 samples per run. Full flowcells can be run usually within two weeks of submission. Partial submissions of less than 7 sample 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 36 base pair read.

Pricing
Priority for Illumina sequencing is currently available for labs associated with the BioMicro Center Core departments. We are able to do Illumina sequencing for other MIT and non-MIT users as space allows on the sequencers. Full pricing information is available at our price list.

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