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SOLiD™ Next Generation Sequencing (NGS)

Applied Biosystems SOLiD™ Next Generation Sequencing platform is a short-read DNA sequencing technology that can produce 60 Gigabases of sequencing data in a single run (V3.0 Plus). Di-base interrogation of DNA templates during sequencing results in high confidence outcomes for variation detection.

Key Technology Features:

  • Ultrathroughput come with data output of more than 60 Gb of data per run (Version 3.0 Plus)
  • High quality 35 to 50 bp reads generated with high confidence di-base encoding
  • Single molecule clonal amplification of templates avoids cloning bias
  • Ability to run up multiple samples on separate channels on two independent flow cells.
  • Fragment library, Mate-Pair library and Paired end library approaches
  • Robust chemistry for accurate base-calling

Applications

Applied Biosystems SOLiD™ Next Generation technologies make possible sequencing projects that may have previously been unfeasible due to cost or technical constraints. The technology can be used for deep sequencing on both RNA and DNA as start material, RNA sequencing is in strand-specific oriented manner.

  • DNA-Seq: Whole Genome Re-sequencing
    • Rapid de novo sequencing and resequencing of genomes
    • Fragment library, Mate-pair library and Sanger hybrid approaches to sequence genomes of any size
  • DNA-Seq: Targeted Genome Sequencing
    • Multiplexing of amplicons and/or patient samples for large SNP detection projects
    • Ultra deep sequencing to detect rare mutations in complex samples
  • DNA-Seq: CHIP-Seq and Methylation-Seq
    • Methods for ChIP-Seq experiments using templates generated by chromatin immunoprecipitation of histone or transcription factor targets
    • High throughput sequencing of bisulfate converted DNA for methylation studies
  • RNA-Seq: Whole Transcriptome Sequencing
    • Affordable reference transcriptome generation
    • Quantification of cDNA expression through SAGE and other tag based approaches
    • Statistically significant numbers of sequenced SAGE tags in a single run without tag concatenation
  • RNA-Seq: Small RNA Analysis
    • Small and micro RNA converted to DNA integrate into Next Gen library preparation protocols for massively parallel sequencing
    • Statistically significant datasets previously unachievable

ncRNA/miRNA Array and Profiling

Oligo Probe Contents

The ncRNA and microRNA custom expression microarray developed in-house with update contents once a year from Sanger miRBase microRNA database. The current MDACC V5.0 contains ~7500 oligo probes designed of ~2000 oligo probes from 695 human miRNA genes encoding 866 active mature miRNAs, 488 mouse miRNA genes encoding 605 mouse active mature miRNAs that sequences collected from update Sanger miRbase (version 12.0). 1200 anti-strand oligo probes of all human and mouse active mature miRNAs are also included. In addition to the probes of miRNAs, 962 oligo probes are designed from 481 human ultraconserved elements, and 3000 probes are designed from specific identified ncRNA in human genome. Positive / negative control oligo probes and miRNA-specific perfect match/mismatch oligo probes as internal control probes are designed and included on array too.

Protocol and Procedure

The oligo probes designed for ncRNA microarray are 40 mer oligo with amine-C6 linker which immobilize covalently to polymer-coated glass-slide surface. To learn more detail of the ncRNA expression profiling procedures, including ncRNA microarray fabrication, sample labeling, array hybridization and signal detection and data analysis, please see Liu et al, microRNA expression profiling using microarray, Nature Protocol, 2008; 3(4):563-578.

miRNA Target Identification and Verification

The current knowledge of miRNA and mRNA interaction is not only at 3’-UTR of mRNA targets, but also 5’-UTR and internal coding region. For mRNA targets interact 3’-UTR with validated miRNAs OmniGrid 100 Arrayer expressed differentially can be predicted using online software of “MIRANDA”, “TARGETSCAN” and “PICTAR-VERT” underneath of specific miRNA interested in Sanger miRbase. The identification of other sites of mRNA targets that miRNA interact with could be predicted by online software “RNA22” or experimentally predicted by using Affymetrix whole genome mRNA expression profiling of the same sample for specific miRNA:mRNA inversely correlation studies based on computing prediction. Furthermore, biological confirmation of the mRNA targets interact with miRNA that expressed differentially by performing Luciferase assay.

Affymetrix GeneChip® System

The  Sequencing and Non-Coding RNA Program supports all Affymetrix GeneChip products and provides full services for all species mRNA expression profiling, SNP genotyping, DNA tiling, CHIP-on chip, and aCGH based on the needs of investigators. All steps of GeneChip analysis are performed according to standard operating procedures (SOP) following the manufacturer's recommendations. For all levels of service including sample preparation and labeling, chip hybridization/staining, chip scanning and initial image analysis are provided and further advanced data analysis is offered in collaboration with statisticians. To ensure reliability of results, continuous quality controls are performed in collaboration with statisticians. These quality controls include:

  1. Testing of reproducibility of sample processing
  2. Analysis of variability of chip production by Affymetrix
  3. Evaluation of reliability of equipment

The average turn-around time is three days or within the same week for small project. The turnaround time for large project depends on the sample number and the queue of samples received in lab. Please visit Affymetrix for updated technology and product availability.

RNA Quality and Quantity Assessment

The same quality and quantity of the start total RNA samples is critical for the comparison of your expression chip data quality. High quality comparable data are generated from equal amount of high quality start material of total RNA.

Agilent Bioanalyzer 2100 is a device of capillary electrophoresis using fluorescence to characterize RNA/DNA size distribution. Bioanalyzer 2100 can measure the Dr. Xiuping LiuRNA/DNA quantity and integrity at nano-and pico-gram levels. High quality of total RNA has clear 28S and 18S ribosomal RNA bands with 28S/18S band intensity ratio of ~2.0 A quantitative measurement for amount and integrity is available. Integrity is quantified via the “Degradation Factor using the "Degradometer software”. To learn more about the technology of BioAnalyzer 2100, please visit: http//www.agilent.com

NanodropND-1000 spectrophotometer enables highly accurate UV/Vis analysis of 1 ul of RNA or DNA protein, dyes samples with remarkable reproducibility. The nanodrop 1000 offers many benefits of small size samples of 1 ul, large dynamic range (2~3700 ng/ul of ds-DNA) without dilution, high absorbance capability, 10 second measurement time, sample is recoverable.

Applied Biosystems 7900HT Real-Time PCR

The Sequencing and Non-Coding RNA Program uses the Applied Biosystems 7900HT Fast Real-Time PCR System. This is high-throughput real-time PCR systems that detect and quantitate nucleic acid sequences. Dual block configurations, the 96-well block and the 384-well block are available.

Key applications include gene expression quantitation (absolute and relative) and the detection of SNPs using the fluorogenic 5' nuclease assay. Both chemistries, the TaqMan™ and SYBR Green, are supported. Today, several companies provide millions of pre-designed, easy-to-use gene expression and SNP genotyping assays for human, mouse, and rat. Applied Biosystems' TaqMan™ MicroRNA Assays and Ambion's mirVana™ qRT-PCR miRNA Detection Kits and Primer Sets are routinely used in lab for the validation of expression profiling data. Northern analysis will eventually be replaced by this method because of the sensitivity and the dynamic range.

Absolute Quantification

This assay is used to quantify unknown samples by interpolating their quantity from a standard curve. As an example, the absolute quantification might be used to correlate viral copy number with a disease state. It is of interest to the researcher to know the exact copy number of the target RNA in a given biological sample in order to monitor the progress of the disease.

Requirements:

  • Standard - any stock DNA or RNA of a known concentration containing the appropriate target in a serial dilution that encompasses the unknown quantities
  • No Template Control (NTC) - a negative control that lacks template
  • Unknown sample – sample's quantity value is unknown but is within the range of given standards

Relative Quantification

This assay is used to analyze changes in gene expression in a given sample relative to another sample, such as an untreated control sample. As an example, relative quantification might be used to measure alterations in gene expression in response to a chemical (drug). Quantification results for the target gene are normalized to a control gene in order to account for sample-to-sample variability; as such, relative quantification does not require equivalent amounts of input cDNA for each sample. In cases where the target and control genes have similar amplification efficiencies, relative levels of target gene expression in samples can be determined without the use of standard curves.

Requirements:

  • Target sequence - sequence that is being studied
  • Calibrator - a sample used as a basis for comparative results (for example, untreated control)
  • Endogenous control - a gene that is present at a consistent expression level in all experimental samples; accounts for sample-to-sample variation with respect to input cDNA
  • Each sample type (for example, each tissue in a study comparing multiple tissues) is amplified by the endogenous control, after which results for the target gene are normalized to the endogenous control results

Calculation methods for relative quantification:

  • Relative Standard Curve Method
  • Comparative Ct Method

Determining Which Method to Use
Both methods yield expression data in terms of sample-to-sample fold changes. However, in order to use the comparative Ct (threshold cycle) method, a validation experiment must be run to show that the efficiencies of the target and endogenous control amplifications are approximately equal.

The advantage of using the comparative Ct method is that the need for a standard curve is eliminated. This increases throughput because wells no longer need to be used for the standard curve samples. It also eliminates the adverse effect of any dilution errors made in creating the standard curve samples.

Multiplexing

To amplify the target and endogenous control in the same tube, limiting primer concentrations must be identified and shown not to affect Ct values. By running the two reactions in the same tube, throughput is increased and the effects of pipetting errors are reduced.

Note: Running the target and endogenous control amplifications in separate tubes using the standard curve method of analysis requires the least amount of optimization and validation.


© 2014 The University of Texas MD Anderson Cancer Center