We offer small and large-scale Sanger DNA sequencing, automated amplicon purification and a suite of next-generation DNA/RNA sequencing technologies. We also are available to provide project consultation and sequencing analysis. We perform and provide state-of-the-art genome and trascriptome sequencing and high-throughput sequencing of antibody receptor and viral genes. Administratively, we provide project consultation and sequencing analysis.
In addition to basic sequencing, extensive analysis of sequence data is also offered. These services include primer design, multiple sequence alignments, construction of phylogenetic trees, determination of genetic distances and mutation rates, recombinant viral genome analysis, and detection of drug resistance mutations.
The Duke Human Vaccine Institute VGA core offers a 3 ways to utilize our facility to accomplish your research objectives.
Sample submission- Researchers provide DNA/RNA or PCR amplicons and service is performed by core staff (Sanger DNA sequencing, PCR amplicon purification and Illumina next-generation DNA/RNA sequencing, HIV co-receptor, subtype and drug resistance analysis).
Independent use- Researchers can reserve and operate the instruments independently and provide all of the required reagents (Illumina Miseq, Illumina NextSeq, real-time qPCR, Bioanalyzer, TapeStation, Qubit fluorometer).
Intermediate use-For Illumina next-generation DNA/RNA sequencing users can prepare libraries and the core will provide appropriate sequencing kits. Core staff will operate the sequencing run. We can also provide library quality control and quantitation.
The first step:
Contact DHVI VGA core manager, Bhavna Hora for consultation and a price quote for your desired service. Our core manager and staff will guide you through the process to achieve your research goal as fast and cost-effective as possible.
The second step:
Register in the CoreResearch@Duke system, which will be used for integrated instrument reservation and billing services. All users must ensure that their NetID's are linked to the appropriate PI and fund codes in the system prior to any work being completed with the BIA Core.
Information on how to establish an account and complete the linking process is available on the CoreResearch@Duke Informational Page, specifically the training guide titled "How to Add a Facility User and Link to a Project".
For Sample Submission, review the Sample Requirements & Submission page.
Large Scale Sanger DNA Sequencing
Equipped with two ABI 3730XL DNA analyzers, the DHVI Viral Genetic Analysis Facility has the daily capacity to sequence up to twenty-four 96-well plates with 95 samples each with reads of up to 1000 base pairs per sample.
PCR Product Purification
The PCR production purification is performed on the Beckman Coulter Biomek FXp Laboratory Automation workstation by utilizing an innovative paramagnetic beads technology for high-throughput purifications of PCR amplicons in which primers, nucleotides, salts, and enzymes in the reaction mixture are efficiently removed.
Detection of low drug resistance mutations
Population sequencing of viral samples to determine the DRMs in the sample
Determination of HIV-1 Co-receptor usage
Our core offers coreceptor usage determination services for NP-2 cells expressing CD4 along with CCR5, CXCR4, or other coreceptors by measuring p24.
Along with sequencing, we also offer:
-Phylogenetic tree analysis
-Calculation of genetic diversity and mutation rate
-Identification of recombinant viral genomes and signature sequence
Amplification of diverse HIV-1 strains
We offer service to design primers and amplify diverse HIV-1 strains as well as other genes
Single Genome Analysis
Our core also offers isolation and analysis of sequences for single nucleotide polymorphisms within the HIV-1 genome.
Illumina MiSeq and NextSeq, Ion Torrent, RNA and DNA Library preparation
We offer services on two Illumina sequencer types: NextSeq and MiSeq. Library preparations can be done either at users end or by the Core staff and used for any of two instruments.
The MiSeq desktop sequencer allows you to access more focused applications such as targeted gene sequencing, metagenomics, small genome sequencing, targeted gene expression, amplicon sequencing starting at 10 ng DNA, and HLA typing. New MiSeq reagents enable up to 15 Gb of output with 25 M sequencing reads and 2x300 bp read lengths.
The NextSeq Series desktop sequencing system provides the flexible power and simplicity you need for transcriptome and targeted resequencing. With tunable output and industry-leading data quality, the NextSeq Series offers the perfect combination of high-throughput sequencing, flexibility and accuracy.
Pooling Samples/Barcoding/Indexing options: For Illumina sequencing, multiple samples can be pooled into an individual lane. Samples may be indexed using Illumina's TruSeq indices, Illumina's Nextera indices or custom indexing adaptors.
Real-time PCR This service is available to quantitate NGS libraries and more. The core houses ABi StepOnePlus™ Real-Time PCR System is a 96-well Real-Time PCR instrument perfect for both first-time and experienced users. The StepOnePlus™ Real-Time PCR System can be setup in a variety of configurations and comes ready to use, out of the box, with intuitive data analysis and instrument control software. Utilizing robust LED based 4-color optical recording, the StepOnePlus™ Real-Time PCR System is designed to deliver precise, quantitative Real-Time PCR results for a variety of genomic research applications.
Sample and library QC
Samples and libarries can we quantified using Nanodrop 1000, Nanodrop 8000, Qubit 3.0 flurometer and Agilent 2200 Tapestation.
The core offers the use of easy-to-use Qubit™ 3.0 Fluorometer and sensitive, specific Qubit™ quantitation assays. Based on the detection of target-specific fluorescence, this integrated system is more sensitive than UV absorbance–based quantification, making it ideal for precious samples and demanding applications.
The Agilent 2200 TapeStation system automates RNA, DNA and protein sample QC, including sample loading, separation, and imaging.
Scalable throughput– Individual channels facilitate variable throughput at a constant cost per sample
Fast – Obtain results in as little as one minute per sample, even for 96 samples.
Easy to use – Simplify your workflow with ready-to-use ScreenTape consumables.
Excellent reproducibility – Achieve user-independent results through minimal manual intervention and pre-packaged reagents.
Our core can generate of single base mutants or multiple base mutants in a plasmid using mutagenesis kits. Primer design is also available. The final clone is sequence confirmed and maxi prepped if needed.
Our core houses 4 Illumina MiSeqs and 1 Illumina NextSeq for High-throughput sequencing of DNA and RNA, and 2 ABI 3730XL DNA analyzers for Sanger DNA sequencing. For quantitative PCR we have an ABI StepOne plus real-time PCR system. Our core also utilizes Biomek FXP robotics for large-scale library preparation and PCR amplicon purification. For DNA/RNA quality control and quantitation we utilize an Agilent bioanalyzer, Agilent Tape station 2200, and a Qubit 3.0 fluorometer.