IDDRC Core - Molecular Genetics

Log In - Sample Submission & Tracking
Log In - GeneSifter Analysis
Description
Services
Instruments
Submitting Samples
Pricing
Software
Frequently Asked Questions

Description:

In November of 2007, the MGCF acquired its first next-generation sequencing platform, the Applied Biosystems SOLiD System Sequencer, through funding from Children’s Hospital Boston and recently upgraded the system to the version 3 platform at the beginning of 2009. Next-Gen sequencing platforms are so named because they are capable of generating billions of bases of sequence per run – magnitudes above the amount generated per the Sanger sequencing reaction method that has been widely used for over a decade. The ability to generate such a vast amount of sequence data in a short amount of time is quickly expanding the way that genomic research is performed and helping investigators discover biological information that was previously unattainable.

The Applied Biosystems SOLiD System Sequencer is unique in its method of performing sequencing reactions. Similar to some of the other technologies, the SOLiD protocol requires an emulsion PCR reaction to amplify each library template. However, during the actual sequence run, the SOLiD instrument utilizes a series of ligation and detection rounds to sequence millions of fragments simultaneously. There are five primer cycles performed on the instrument with each cycle staggered by a single base and including a series of seven or ten ligations for either a 35 or 50 base pair sequencing run. Each ligation decodes two bases and is recorded through fluorescent imaging. By compiling the fluorescent reads in color space for each fragment, an accurate sequence can be generated. A much more detailed description of the technology including videos, literature, and publications is available at the Applied Biosystems SOLiD System Sequencing page.

Services:

The Next-Gen Sequencing Facility is currently preparing SOLiD instrument runs for full slides on fragment libraries. Fragment libraries include whole genome resequencing, targeted resequencing, RNA-seq, ChIP-seq, and small RNA libraries. Customers may create their own libraries after project consultation with the Core and submit them for quantitation and processing. Library preparation is also available in the Core for whole genome, whole transcriptome, and small RNA fragment libraries.

A full slide instrument run consists of all post-library construction processing through to providing sequence data as follows. The Core performs a quantitative PCR (qPCR) assay on every library to determine its concentration. Using the information from the qPCR, the Core performs a series of emulsion PCRs (ePCR) to amplify template onto beads with individual fragments from the library (or pool of bar-coded libraries). At this stage a spike-in control library is added for processing validation once the run is complete. The completed ePCR then undergoes a breaking and enrichment procedure to release the templated beads from the oil mixture and to enrich for the templated beads that will be deposited onto slides to run in the SOLiD instrument. Before a full run is conducted in the SOLiD, a quality control run is performed to ensure that the ePCR and breaking worked properly. Once the beads pass the QC run, a full slide run is performed on the instrument. Upon completion, each customer is provided with the color space sequence data and run quality data. The Core will keep a record of this data as well as the raw intensity files in permanent electronic records, however the intensity files are not necessary for downstream analysis. All image data is discarded once the sequence run alignment is confirmed by analyzing our spike-in control library data.

As of July 2009, the MGCF has teamed up with Geospiza to offer SOLiD sequencing bioinformatics support through their GeneSifter on-line application. Use of this software is included in the cost of an instrument run for up to three months after data is posted. This software allows users to perform alignments to their reference species as well as perform downstream data analysis. Currently, analysis pipelines are available for RNA-seq, Digital Gene Expression, and small RNA SOLiD sequencing runs, but many more analysis pipelines are under construction and will be available soon. For more information about using the GeneSifter application through the Next-Gen Sequencing Facility, please email genome@dnacore.org.

Over the next few months, the Core will be working on preparing and running mate-paired libraries and testing out various methods for generating targeted genomic libraries. To discuss SOLiD sequencing projects, please contact the Core at genome@dnacore.org.

Instruments:

Applied Biosystems SOLiD System Sequencer
Covaris S2 Sonicator
Applied Biosystems 9700 Thermal Cycler with dual 96-well blocks (4 each)
Agilent 2100 Bioanalyzer

Submitting Samples:

To begin a Next-Gen sequencing project with the core, please contact genome@dnacore.org to arrange a consultation. Once project details have been coordinated, an account is created on our on-line Next-Gen Sequencing tracking system and samples can be submitted to the Core.

Template Requirements for Library Preparation:
Fragment Genomic DNA Library - 2.5 ug genomic DNA
Small RNA Library - 2.5 ug DNase-treated total RNA or 500 ng DNase-treated miRNA
Whole-Transcriptome Library - 200 ug DNase treated total RNA for a Poly-A purified RNA library using the Invitrogen Poly-A Purist kit or 2.5 ug DNase-treated total RNA for a ribosomal RNA depleted library using two rounds of purification with the Invitrogen Ribominus kits.

Pricing:

SOLiD sequencing charges are divided into the various stages of template preparation. Library construction is the first phase, followed by library quantitation, templated bead preparation, and a single full flowcell run on the SOLiD System Sequencer. Libraries may be bar-coded and combined to multiplex samples for templated bead prep and instrument runs on a single flowcell. Multiple ePCRs are generally required to generate an appropriate amount of quality templated beads for a single run. All library construction and library quantitation prices are posted as a per library charge.

Due to the massive amount of data generated on the SOLiD instrument, there is also a separate data storage charge for users who wish to store their intensity data files permanently on the Next-Gen Facility servers. This fee is a one-time charge per flowcell run on the instrument. All the csFASTA, qual, and stats files generated with every run are automatically stored at no extra fee. All of our data is stored on location on an arrayed server with built-in redundancy for single drive failures and this server is also mirrored off-site at a data center.

For more information about our data storage or any of our other services, please feel free to contact genome@dnacore.org.

To help assess complete project pricing, an example of a typical full flowcell charge for a 50 bp sequencing run including all preparation post-library construction is as follows:
10 bar-coded libraries ($25 x 10) + 5 ePCRs ($500 x 5) + 1 flowcell run ($11000) = $13750.

Software:

Geospiza GeneSifter Analysis - Offered through the Core for up to three months for every run. Accounts are created upon completion of a run.
Applied Biosystems SOLiD Software Community – a SOLiD data analysis resource for scientific researchers and analysis software developers.