Services and Fees

Illumina Next Generation Sequencing

Overview

The DNA Analysis Facility now offers next generation sequencing  (NGS) on the Illumina GAIIx and the Illumina HiSeq2000 platforms.

Illumina HiSeq2000 Next Generation Sequencing - NEW SERVICE

The HiSeq2000 is Illumina’s newest and most advanced sequencing platform. Like the GAIIx, the HiSeq2000 operates on Illumina’s well-established reversible terminator-based sequencing by synthesis chemistry. It is capable of generating more than 500 gigabases (typically 500 Gb-550 Gb) of sequence per paired-end instrument run with read lengths of 2X100bp. Upgrades to the flow cell, imaging software and reagent chemistry are forecasted to increase instrument output to one terabase per run by December 2011.

The Technology and Workflow

The HiSeq2000 workflow can be divided into four parts: library preparation, cluster generation, sequencing by synthesis and data analysis. 

Library Preparation

A NGS library is made up of random fragments that represent the entire sample. It is created by shearing DNA (Covaris S220) into 150-250 base fragments. These fragments are ligated to specific adapters then enriched by PCR amplification. The resultant library is checked for quality and quantified.

Cluster Generation

Library fragments are bound to a flow cell by hybridizing the fragments to a lawn of oligonucleotides complementary to the adapter sequences. Bound fragments are clonally amplified by bridge amplification to create millions of individual dense clusters of clones. Each cluster contains approximately 1000 clones.

HiSeq2000 Sequencing 

Sequencing of the flow cell employs Illumina’s well-established sequencing by synthesis chemistry. This chemistry utilizes four reversible terminator nucleotides, each possessing a different fluorescent dye and a chemically blocked hydroxyl group. During sequencing, fluorescently labeled nucleotides are flowed across the flow cell. A single nucleotide is incorporated into each DNA, resulting in the simultaneous one base extension of millions of DNA clusters. The incorporated fluorescent nucleotides are excited by a laser, and emit their characteristic fluorescence. The fluorescence is detected and recorded in an imaging step. Following base detection the fluorescent dye is enzymatically removed and the blocking group is chemically removed from the incorporated nucleotide to allow incorporation of the next nucleotide. This is repeated 36 to 100 times, generating a series images.

Data Analysis

The raw data generated is imaged, and basecalled before sequence analysis begins. Data analysis is performed in collaboration with faculty from the department of bioinformatics.

Paired End Runs

The paired end module is used to perform sequencing from both ends of the adapter-ligated fragments. Using the paired end module fragments are first sequenced from one end then essentially flipped, and sequenced from the other direction. This doubles the amount of sequence data obtained from each cluster and may provide positional information.

Services Provided

The Illumina GAIIx and Hiseq2000 sequencers are very flexible platforms, enabling a wide variety of applications that differ only in sample preparation and downstream data analysis.

Run options include: 36, 54, 72 and 100 base single read or paired end.

Services Include:

ChIP-seq

To identify specific transcription factor (protein) binding sites in specific tissue and cell types; also to identify the occupancy of specific chromatin proteins and specific histone modifications throughout the genome.

RNA-seq

• Quantitative and/or Qualitative
• Quantitative Gene Expression Analysis: determines level of RNA expression by counting number of transcripts of individual genes throughout the genome
• Qualitative- For the identification of:
  • novel transcripts
  • alternative splice site
  • rare transcripts
  • coding SNPs

Exome Resequencing

• The Human Genome is approx. 3 billion bp; 1.2%-1.6% coding
• Exome sequencing selectively enriches for and sequences coding regions
• Exome sequencing is used for:
  • The identification of causative mutations in genetic disorders
  • The discovery of functional variations for traits
  • The discovery of functional variations for drug responses

Targeted Resequencing

• Selectively enriches for and sequences user defined regions/targets of interest

Methylation Sequencing

• Genome-wide methylation analysis
• Sequence bisulfite treated genomic DNA
• Detects variations in methylation signatures with single-base resolution

Whole Genome Sequencing

• Human, Mouse, Rat, Yeast Monkey, Viral, Bacterial plus other genomes
• Cancer research- sequence matched tumor/ normal samples
• Provides information on copy number and structural variation

Getting Started

First-time next generation sequencing service users and investigators with large-scale projects are required to attend an initial consultation meeting with DAF NGS specialist and bioinformaticians. To schedule a consultation please contact Erika Thompson, ejthomps@mdanderson.org.
Contact Dr. Louis Ramagli or Dr. Hongli Tang to schedule a time to drop off samples. Sample(s) must be submitted with a sample sheet.

HiSeq2000 Submission Form (doc)

Sample Requirements

Sample Preparation Services

HiSeq2000 Sequencing Cost

Estimate only; sequence output is not guaranteed.
Note: Library preparation is not included in the sequencing price.

Multiplex samples using barcodes

To reduce cost, we are using barcodes that identify individual samples, which are then mixed together for sequencing in a single lane.
Multiplex:

• 3+exomes/lane (30x-40x coverage)
• 4-6+ ChIP-seq/lane
• 4+RNA-seq/lane

GAIIx Sequencing Cost

*Note: GAIIx sequencing cost includes one library preparation per lane.

DNA Analysis

Bioinformatics Support

Bioinformatics support is provided in collaboration with faculty from the Department of Bioinformatics.

Bioinformatics  Faculty Collaborators

• ChIP-Seq: Shoudan Liang, Ph.D.
• All other services: Xiaoping Su, Ph.D., Jack Zhang, Ph.D.

Sanger Based DNA Sequencing

DNA sequencing services are performed by experienced molecular biologists with the goal of providing high quality DNA sequencing data. The DAF offers DNA sequencing from single or double stranded DNA templates. ABI Big Dye terminator cycle sequencing chemistry is used for the majority of our sequencing reactions. Sequence read-lengths are normally between 800 and 900 bases with a base-calling accuracy >98.5%. The sequence reliability can be increased to 99.99% by sequencing the same template in the opposite direction. The facility quantifies all samples, performs sequencing reactions and cleanup, collects data by capillary electrophoresis, analyzes the data, and provides sequence as text files and chromatograms.  Sequence chromatograms and the corresponding text files are deposited on the DAF server. All data is password protected.

The typical turnaround time is one to two business days.  Longer turnaround times may occur when our sample volume is very high and when we experience instrument problems.

Genetic Fragment Analysis

Automated genetic fragment analysis can be used to study genotypes, loss of heterozygosity and microsatellite instability. This technique involves using fluorescent primers during amplification to label PCR products. These products are then separated on a capillary platform. Consultations should be arranged with Erika Thompson at 713-834-6381 or ejthomps@mdanderson.org before projects are initiated. Users are responsible for providing fluorescently labeled PCR products. These will be run together with sizing standards on the 3730 genetic analyzer.

Taqman Based Gene Expression Analysis

The DAF performs commercially available gene expression assays on the RNA provided by the investigator and will custom design assays at the investigators request. This service includes RNA quantification, RNA QC, reverse transcription, DNase1 treatment and TaqMan® PCR based quantitative gene expression assay setup and analysis. MicroRNA assays are included in this service. In addition to the standard Gene Expression Analysis Service the DAF provides investigators with the option of running Low Density Array cards. These customized 384-well micro fluidic cards allow investigators to analyze gene expression patterns in many samples across a defined set of gene targets. Eight samples can be assayed for as many as 48 gene targets simultaneously.

* $200-$250 One-time setup fee 

Resequencing: Whole Gene Mutation Detection

Gene resequencing provides a fast, simple method of detecting variants in cancer related genes. It allows an entire gene to be evaluated in a single experiment. The DAF performs this assay using Applied Biosystems VariantSEQr^® kits, along with our own custom designed primers. The DAF will custom design and optimize resequencing for any human gene requested by an investigator. Please contact the DAF for the most current list of genes available. Cost depends on gene sequence complexity and the number of amplicons/ sequences necessary to cover the regions of interest. Please contact the DAF for specific project pricing.

Resequencing Gene List

Resequencing Service Includes:

• Quantitation and normalization of samples
• PCR amplification and purification
• Sanger sequencing (AB3730XL)-both directions
• Comparative alignment to reference sequence (SeqScape software – Applied Biosystems)
• Reanalysis (if needed)
• Report of all mutations found and all sequences generated

Pricing