Viral / Molecular Core High Density Sequencing Support
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.
- Initial consultation on experimental design (free of charge)
- Deep sequencing of HIV-1 viral genomes
- Deep sequencing of bacterial 16S rRNA gene sequence tags
- Programmer time for data analysis
- Optimized bar-coded primers for amplification
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
Order High Density Sequencing services through the Path BioResource at Penn. Instructions for the Path BioResource can be found here on the CFAR website or on the Path BioResource site itself.
Core Service Contact:
Frederic Bushman, Ph.D.
Core Co-Director, Viral and Molecular Core
The goal of these studies is to obtain deep sequence information on bacterial populations as reported by 16S rRNA gene sequencing of the V1V2 region. Up to 46 samples can be analyzed per sequence run. Typical applications might be tracking differences in bacterial populations between sick and healthy populations or between body sites. Bar coded primers are used to amplify the 16S rRNA gene V1V2 region over many samples (PMID:17576693). Amplicons are pooled, and the subjected to 454/Roche pyrosequencing. Sequence reads are then 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. Samples are analyzed for within sample diversity and between sample diversity, and clustering versus user-defined metadata. Statistical tests are also included at no extra charge, mostly using the UniFrac distance metric. 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).