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Tulane Center for Aging COBRE Grant Genomics, Biostatistics, and Bioinformatics Core

 

The Genomics, Biostatistics, and Bioinformatics Core at the Tulane Center for Aging provides the following services:

  • Next-generation sequencing (NGS), with the Ion S5 XL system (Ion Torrent/Life Technologies).
  • DNA Sanger sequencing, with the 3130xl Genetic Analyzer (Applied Biosystems).
  • RNA and DNA sizing and quality determination, with the 2100 Bioanalyzer (Agilent).
  • Readings for luciferase reporter assays, with the Modulus Microplate Luminometer (Turner). 
  • DNA quantification, with the XPS Fluorescence Microplate Reader (Gemini).
  • Bioinformatics and biostatistics support is available for analysis of the data acquired through the core facility and COBRE projects, including NGS data analysis.
  • Cell line validation with the AmpFLSTR Identifiler Plus PCR Kit. (New Service)

Other equipment available for use:

  • QuantStudio 7 Flex Real-Time PCR System is available for gene expression analysis (Life Technologies).
  • Attune® Acoustic Focusing Flow Cytometer (Life Technologies).

The core can also help project investigators with:

  • Refinement of experimental design, including sample size & power.
  • Determination of adequate control groups, etc.
  • Development (and implementation) of appropriate analytical plans to test hypotheses derived from the specific aims of each project.
  • Interpretation and presentation of results from analyses.

Investigators can discuss applications and obtain information about the time-frame for services.

Next-Generation Sequencing
Ion S5 XL, AB Library Builder, and Ion Chef System

Next-generation sequencing (NGS) is a technology for high throughput and high depth sequencing. There are several applications involved: whole-genome sequencing (WGS), whole-exome sequencing (WES), transcriptome analysis (RNA-Seq or AmpliSeq) and others.

The Ion S5 XL (Fig. 1) next-generation sequencing system, funded by COBRE project, utilizes semiconductor technology based on direct real-time measurements of hydrogen ions (protons) liberated as a result of phosphodiester bond formation during DNA replication.

Ion S5 XL System

Fig. 1. Ion S5 XL system

Recently paired with Ion S5 XL, the Ion Chef (Fig. 2) and AB Library Builder (Fig. 3) significantly simplify the next-generation sequencing workflow with automating library preparation, template generation, and chip loading. This modification increased the volume of our experiments (RNA-Seq, AmpliSeq, miRNA-Seq and whole-exome sequencing).

Fig. 2 Ion Chef System
Fig. 2. Ion Chef System

AB Library Builder
Fig. 3. AB Library Builder

 

Service

WES

WES

RNA - Seq

RNA - Seq

miRNA - Seq

Coverage

130 - 160X

60 - 75X

~20M reads

~40M reads

Depends on source

 

Result Format

Data from exome sequencing are generated in BAM format. Data from RNA-Seq are generated in FASTQ format. NGS files are saved on an external drive and provided by the core to investigators. Other results will be provided as Excel or pdf files or other readily accessible formats and sent via e-mail to investigators with source files available on request.

Sanger Sequencing
3130xl Genetic Analyzer

The Applied Biosystems 3130xl Genetic Analyzer (Fig. 4) is a 16-capillary electrophoresis instrument for Sanger sequencing with read lengths up to 900 bps. This system is useful for a wide variety of sequencing applications: SNP validation, SNP screening, mutational profiling, cell line authentication with the AmpFLSTR Identifiler Plus PCR Kit, etc.

3130xl Genetic Analyzer

Fig. 4. 3130xl Genetic Analyzer

Human Cell Line Authentication Service

Misidentification and cross-contamination of cell lines is a recurrent problem in life sciences research. In response, various scientific journals and funding agencies are enforcing strict usage of well-identified cell lines for research purposes and that cell line authentication must be performed prior to grant approval and/or publication. We offer comprehensive Human Cell Line Authentication Services using Short Tandem Repeat Profiling (STR) technology with AmpFLSTR Identifiler Plus PCR Kit (Life Technologies). This kit uses 16 markers in the human genome (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, Amelogenin, D5S818, and FGA) to ensure the correct identity of your cells, avoiding the risk of misidentified or contaminated cell lines in your research. 

Agilent 2100 Bioanalyzer

The Agilent 2100 Bioanalyzer (Fig. 5) provides highly reproducible on-chip electrophoresis that allows sizing, quantitation and quality control of DNA and RNA. It is used extensively in the NGS workflow for quality control and provides quality determination of RNA samples prior to RNA-seq. 

Agilent 2100 Bioanalyzer

Fig. 5. Agilent 2100 Bioanalyzer

QuantStudio 7 Flex Real-Time PCR System

The Applied Biosystems™ QuantStudio™ 7 Flex Real-Time PCR System (Fig. 6) delivers the proven reliability, sensitivity, and accuracy of the data in a new industrial design. The QuantStudio 7 Flex System has been optimized to enable the broadest range of quantitative PCR applications, with additional dyes, formats, and automation options. This systems allows researchers to use TaqMan Array cards, running up to 384 simultaneous real-time PCR reactions by simply loading the card with your sample and centrifuging, producing consistent results with less pipetting error.

QuantStudio 7 Flex Real-Time PCR System

Fig. 6. QuantStudio 7 Flex Real-Time PCR System

Modulus™ Microplate Luminometer

This system is very sensitive: the luminescence channel is separated and positioned directly above the sample well, maximizing light capture for the best possible sensitivity. The Modulus™ Microplate Luminometer (Fig. 7) is between 10 and 1000 times more sensitive than other luminometers, simultaneously measuring samples of varying brightness. It is also designed to reduce crosstalk to achieve this extra-large reading range.

Modulus Microplate Luminometer

Fig. 7. Modulus™ Microplate Luminometer

Attune Acoustic Focusing Flow Cytometer

The Attune Acoustic Focusing Flow Cytometer (Fig. 8) is the first cytometer that uses ultrasonic waves (similar to those used in medical imaging), rather than hydrodynamic forces, to position cells into a single, focused line along the central axis of a capillary. This cytometer is used for immunophenotyping, cell cycle analysis, cell proliferation, apoptosis, GFP & RFP detection, microbiological projects, and a broad range of other cell biology applications. Data obtained with and acoustic flow cytometer are more precise (narrower fluorescence peaks) than data obtained with traditional hydrodynamic focusing instruments, resulting in greater separation and easier data interpretation. The instrument has a violet/blue laser configuration: 405 nm (50 mW) and 488 nm (20 mW) lasers, with three emission channels per laser.

Attune Acoustic Focusing Flow Cytometer

Fig. 8. Attune Acoustic Focusing Flow Cytometer

 

Location and Contact Information

Genomics, Biostatistics, and Bioinformatics Core
School of Medicine
6th floor
Room 6304
1430 Tulane Avenue
New Orleans, LA 70112

Phone: 504-988-3090

E-mailmczarnyr@tulane.edu 

 

Acknowledgements

The COBRE Genomics, Biostatistics, and Bioinformatics Core at the Tulane Center for Aging, Tulane School of Medicine is supported by NIH: NIGSM COBRE grant No. P20GM103629 to Dr. S. Michal Jazwinski.

The Ion Chef was funded in 2016 by Core Lab Equipment Award from the Tulane School of Medicine.