SOLICITATION NOTICE
68 -- Library Construction, Genome Sequencing & Finishing
- Notice Date
- 9/4/2003
- Notice Type
- Solicitation Notice
- Contracting Office
- Department of Health and Human Services, National Institutes of Health, National Institute of Allergy & Infectious Diseases/AMOB, 10401 Fernwood Drive, Suite 2NE70, MSC 4811, Bethesda, MD, 20817
- ZIP Code
- 20817
- Solicitation Number
- RML-RFQ-3049
- Response Due
- 9/19/2003
- Archive Date
- 10/4/2003
- Point of Contact
- Julienne Keiser, Purchasing Agent, Phone 406-363-9370, Fax 406-363-9376, - Rebecca Guenthner, Chief Contracting Officer, Phone 301-402-2284, Fax 301-480-3695,
- E-Mail Address
-
Jkeiser@niaid.nih.gov, rguenthner@niaid.nih.gov
- Small Business Set-Aside
- Total Small Business
- Description
- This notice is a combined synopsis/solicitation for commercial items prepared in accordance with format in Subpart 12.6, as supplemented with additional information included in this notice. This announcement constitutes the only solicitation; quotes being requested & a written solicitation will not be issued. This procurement is being issued as a request for quotation. Submit offers on RML-RFQ-3049. Solicitation documents and incorporated provisions & clauses are those in effect through Federal Acquisition Circular 2001-15 dated 8/25/03. This acquisition will be processed under Simplified Acquisition Procedures (SAP) and is a Small Business Set-Aside. The North American Industry Classification System (NAICS) code for this procurement is 325414 & the small business size is 500. SCHEDULE: Laboratory of Human Bacterial Pathogenesis (LHBP) has a need to sequence to 10X coverage a Mycobacterium Tuberculosis genome and to sequence to 7X coverage a Streptococcus Pyogenes genome, involving the following three steps: One(1) Library construction, Two(2) 10X and 7X shotgun sequencing, and Three(3) Two(2) rounds of finishing (primer design, synthesis, and sequencing) for each genome. The Contractor shall accomplish 7X and 10X sequence coverage of two bacterial genomes. Personnel suggested by the Contractor will be approved in advance by the RML Project Officer. This is an eight(8) to ten(10) month work effort, where the final product will be the production of two(2) genomes, at 7X and 10X coverage, with two(2) rounds of finishing performed on each. The trace files must be compatible, transferable to, and readable by RML bioinformatics software and RML computing systems and infrastructure, which involves a Finch, and ERGO server. RML systems are PC, MAC and Unix/Linus-based. The contractor shall keep all NIH, RML-derived data secure and will not release, make public or maintain any of the data beyond this contract effort. If Internet connectivity is needed in order to perform the work, for transfer of files, or other efforts, contractor computers will be secure and in compliance with RML-IT security procedures and policies. Contractor-supplied software shall be kept current and up-to-date with applicable security patches and upgrades. Internet connectivity and/or remote access for the purpose of performing the work, for transfer of files, or other efforts, will be coordinated with NIAID IT staff to ensure security and compliance with NIAID IT procedures and policies, including Section 508 Accessibility Standards. The Contractor shall provide the following three(3) steps: (1) Library construction. The MTB genome is estimated to be approximately ~4.4 MB in size (4,411,529 base pairs for strain H37Rv). The Streptococcus genome is estimated to be approximately 2.0MB in size. The LHBP will send to the contractor purified total genomic DNA for these two(2) strains. The contractor shall use this DNA to construct a library using the plasmid vector pGEM-3Z. DNA shall be fragmented using a computer controlled shearing device to achieve an average DNA fragment size of 2 kb. The fragments shall be treated with T4 polymerase followed by Klenow fragment before ligation into the plasmid vector. Ninety percent or greater cloning success of 2kb fragment representation in the clones shall be achieved. Progressive assemblies of the incoming data shall begin after the first 1,000 clones from the library have been sequenced. This shall be done to detect any problems in library construction or contamination from other organisms/vectors. The assembly dynamics shall be carefully compared with models generated on both real data and computer simulations. These tests can reveal irregularities at many levels of the process, especially those involving library insert size distribution. (2) 10X shotgun sequencing/preliminary annotation/comparison. Transformed colonies shall first be grown in petri trays and picked by a robotic colony picker. Once colonies are picked, each clone must receive an individual name that must reflect the organism, the type of the library, the cloning attempt, the ID of the 96- well plate and it’s relative position in the plate. Cultures shall be processed for DNA mini-preps using robots for plasmid extraction and purification steps. 96 well plates with purified plasmid DNAs must be stored in dedicated, specific freezers at -20 Celsius. DNA samples shall be transferred to 384 well plates for performing sequencing reactions. IDs of the 96- well plates must transfer to the 384-well format for tracking. Sequencing reactions shall be performed using the ET MegaBACE Dye Terminator Kit. Ethanol precipitation may be used for PCR clean-up. Sequencing reactions may be loaded onto MegaBACE 1000 DNA sequencers or #3700 DNA sequencers. Samples shall be sequenced from both ends using standard forward and reverse, vector-specific primers for both plasmid libraries. Passing sequence reactions shall be those at 450 bases of average Q19-20 level or higher. A minimum of approximately 98,000 sequencing reactions at average 450 bases length or higher, and average Q19-20 quality or higher, shall be performed for the MTB genome. A minimum of approximately 32,000 sequencing reactions at average 450 bases length or higher, and average Q19-20 quality or higher, shall be performed for the Strept. genome. No more than 98,000 or 32,000 sequencing reactions for each genome may be performed without written approval by the project officer. Following 10X shotgun sequencing of a genome the assembly should produce between 200 and 300 contigs or gaps per genome. The contractor shall compute, based on the assembled sequences and by summing the sizes of the contigs, a better estimate of the genome sizes. The contractor shall do any extra runs needed to bring the average coverage to 10X. Actual payment shall reflect the original runs plus any extra runs made to bring the coverage to 10X. If the original runs indicate a smaller number of runs would have been adequate to achieve 10X coverage, LHBP will pay for the actual runs done. LHBP recognizes that genome size is an unknown and may vary per strain. If the total number of runs brings the coverage to slightly greater than 10X, this extra coverage will be useful to LHBP. Primary assembly of the individual sequences shall be performed using the Phred/Cross_match/Phrap package originally developed at Washington University. These algorithms automatically base call the trace data, screen out vector sequence, remove poor data, and assemble individual reads into contigs. The contractor shall have additional in-house tools to help in the preparation of the data for assemblies, to modify the assembler parameters, assembler output, to add and remove data from the assembly and to compare different or sequential assembly runs. The assembly dynamics shall be monitored and compared with models generated on both real data and computer simulations to reveal potential problems associated with the assembly conditions. The integrity of the assemblies shall be checked using either in-house or commercial tools, to detect potential errors and ambiguities in contigs. The contractor shall be able to adjust assembly conditions and perform manual editing to correct some of these types of problems. 10X sequencing reads and assemblies shall be delivered and must be compatible with our Finch server. (3) Two rounds of genome finishing. Two(2) rounds of primer design and synthesis for use in sequence walking reactions using the plasmid library shall be performed using Consed/AutoFinish editing software. Various amounts of manual checking and correction may be required, depending on genome-specific factors (GC%), repeat composition, library construction, etc. Additional sequencing in the two rounds of finishing shall be used to raise quality levels for problem regions and to join contigs. Deliverables:
- Web Link
-
Link to FedBizOpps document.
(http://www.eps.gov/spg/HHS/NIH/AMOB/RML-RFQ-3049/listing.html)
- Place of Performance
- Address: Rocky Mountain Labortories 903 S. 4th Street Hamilton, MT
- Zip Code: 59840
- Country: USA
- Zip Code: 59840
- Record
- SN00426845-F 20030906/030904214332 (fbodaily.com)
- Source
-
FedBizOpps.gov Link to This Notice
(may not be valid after Archive Date)
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