Overview

The Genomics Core Facility performs next-generation sequencing using the Life Technologies SOLiD system 5500xl. Next-generation sequencing provides a broad range of applications including:

Genome

• De Novo Sequencing
• Targeted Resequencing
• Whole Genome Resequencing

Epigenome

• Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
• Methylation Analysis

Transcriptome

• Gene Expression Profiling
• Small RNA Analysis
• Whole Transcriptome Analysis

In contrast to microarray, the sensitivity is much greater and discovery of the unknown is possible. With rapid technological improvement, the cost of next-generation sequencing experiments is within the reach of most researchers.

Next-Generation Sequencing Facts

• SAGE sequencing for measurement of digital gene expression can be performed less expensively than microarray gene expression while providing expression data for known and unknown transcripts with 100 fold greater sensitivity.
• The inherent error checking properties of two-base encoding and a reduction in raw error rates provide highly accurate data with more than 80% of bases having quality values of ≥ 30
• Greater than 99.94% base-calling accuracy due to 2-base encoding
• Greater than 99.999% consensus base call accuracy at 15 times fragment coverage of the sequence
• Samples may be multiplexed
• Transcripts have been detected at levels less than 1 copy per cell to over 100,000 copies per cell for a dynamic range of 10⁵.
• A comparison of dynamic range: Microarray 2-3 logs, SOLiD 5 logs, qPCR 7-8 logs.

Genomics Core Facility Next-Gen Sequencing Service

If you are planning to use our next-generation sequencing service, please contact us as early as possible in your grant proposal or experiment planning process. We will discuss the details of your proposal to determine the optimal experimental strategy, scheduling, and estimated cost.

The GCF provides:

• Information and support for grant submission
• Experiment design consultation
• Application specific library preparation for DNA or RNA libraries
• Preparation of library samples for the SOLiD
• SOLiD sequencing
• Primary and secondary data analysis including base calling, mapping to a reference sequence, and limited application specific tertiary analysis (digital gene expression, clustering etc.)

Experiment Example

1. Total RNA with the small RNA fraction is isolated using the mirVana Kit (column purification).
2. The small RNA is separated using FlashPage.
3. Sequencing adapters and bar-codes are ligated to the small RNA fragments.
4. Emulsion PCR attaches the fragments to beads for sequencing.
5. Sequencing by ligation on AB SOLiD.
6. On-instrument primary analysis then data is uploaded to Geospiza GeneSifter for storage, alignment to a reference genome, fragment counting, and statistical analysis.

Two data sets and pathway analysis are merged to create a biological story. In this experiment, known genes of interest from microarray analysis of mRNA transcripts are cross-referenced with microRNA predicted to interact with them. Thus regulation effects can be easily seen. Groups of regulated genes are submitted for pathway analysis to determine biological effect. This is an example of a small activating RNA (saRNA).

Links

• Applied Biosystems SOLiD System
• Learn the basics of SOLiD Sequencing Chemistry
• Learn about SOLiD System accuracy
• SOLiD Community