- Shotgun Library Construction and Sequencing
- 454 Sequencingâ„¢ System
MilleGen provides 2 complementary approaches, shotgun and 454 technology (GS-FLX) to sequence a variety of samples including microbial and fungal genome, BAC, PAC, or cosmid DNA.
The number of sequenced clone (shot gun) or 454 run will depend on the requested coverage.
The 454 GS FLX system allows to obtain at least 80 Mb of data per run. A 20-30 X genome coverage provides more than 95% of the sequence.
Examples: (data obtained with a GS 20 from 454 Life Science)
- 3 runs of E coli genome (4.6 Mb) allowed to obtain 97 % of the total sequence (180 contigs)
- 3 runs of B. licheniformis genome (4.2 Mb) allowed to obtain 97 % of the total sequence (124 contigs)
- 20 runs of Neurospora crassa (40 Mb genome): 7895 contigs
Shot gun service includes:
DNA isolation (genomic DNA, BAC…)
Shotgun library construction by shearing of the DNA sources
Size selection of fragmented DNA (1-3 kb)
Subsequent subcloning of fragments into a cloning vector
Library quality control check. E-coli genomic DNA contamination and empty vector contamination by cross-match. Less than 5% contamination will be accepted.
Plasmid purification of selected clones
Primer extensions with forward and reverse primers
Quality and vector trimming of the sequences
Assembly of the overlapping sequences
Closing of the remaining gaps by primer walking until a single contig is reached (optional)
