BioMicroCenter:Small RNA Seq

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Samll 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. 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. If this is a service that you would like the BMC to perform in the future please visit the Ideas page. Illumina DGE-small RNA sample preparation kits can be purchased through the BioMicro Center for $1,800.00 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.

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 known genomic loci's of all miRNA looking for an exact match. 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.

Pricing

Illumina sequencing is currently available only for labs associated with the BioMicro Center Core departments.

ILLUMINA SEQUENCING	CORE LAB/MIT	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.
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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