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Penn Center for AIDS Research

Viral / Molecular Core High Density Sequencing Support

Core Services Main Page | Virology | Molecular | Sequencing | SGA Services | Intergrated vDNA | Other

High density sequencing is an emerging technology with the capacity to generate millions of sequences, with up to a billion bases of sequence in a single run. This approach is ideal for application to complex genetic populations such as HIV and SIV, HBV/HCV, complex bacterial populations and other applications.

The Viral/Molecular Core is now offering 454/Roche pyrosequencing as a service. In each case, amplicons will be generated from samples of interest, then sample sequences determined. Runs typically yield ~100,000 sequence reads of >250 bp with good quality scores. Runs are typically divided up among 46 bar-coded individual samples, but can be utilized for fewer samples if greater depth is needed. Sequences are subject to stringent quality control algorithim and provided to users in fasta format. The core also provides advice on experimental design, sequence work up, statistical analysis, and long term data storage.

Several services are offered.

  1. Initial consultation on experimental design (free of charge)
  2. Deep sequencing of HIV-1 viral genomes
  3. Deep sequencing of bacterial 16S rRNA gene sequence tags
  4. Programmer time for data analysis
  5. Optimized bar-coded primers for amplification

Pricing

The Core provides support for each of these areas thought the expertise of Dr. Rick Bushman (Core Co-Director). Advanced bioanalytic support and analysis is offered collaboratively through the Biostatistics and Data Management Core (Core G) by Dr. Hongzhe Li (Core G Co-Investigator).

For further information on utilizing V/M Core support for high density sequencing, contact Dr. Rick Bushman.

Core Service Contact:

Frederic Bushman, Ph.D.
Core Co-Director, Viral and Molecular Core
Phone: 215-573-8732
Email: bushman@mail.med.upenn.edu


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Deep sequencing of HIV-1 viral genomes

Deep SequencingThe goal of these studies is to obtain deep sequence information on specific regions of a virus of interest. Up to 46 samples can be analyzed per sequence run (average 105 sequences), though fewer samples may be run if specific applications require greater depth of sequencing. Typical applications might be tracking development of drug resistant or immune escape variants over time. Bar coded primers are used to amplify the same region over many samples (PMID:17576693). Amplicons are pooled, and the subjected to 454/Roche pyrosequencing. Sequence reads are then quality filtered, separated based on bar codes into the original samples. Initial sequence work up, analysis, and storage are included in the price of the sequencing run. Analysis may include quantification of diversity within each sample, or tracking one or several user-defined sequences across the sequence population; additional forms of basic analysis may be included in the base cost upon discussion with Core personnel. More sophisticated analysis can be purchased via programmer time (item 4, above).

Examples of analysis can be found at PMID:18614863, PMID:17576693, PMID:20375170).