Microbial Finishing at DOE Joint Genome Institute (JGI): Sequencing Difficult DNA Templates
The US DOE Joint Genome Institute (JGI) mission is to provide the scientific community with high-quality finished genomes. Approximately 300 microbial genomes are currently in the JGI pipeline and to date, 65 have been completed. The objective of the Microbial Finishing laboratory is to process sequencing reactions in order to close physical gaps, sequence gaps, and to increase quality of reads. Since most of the genomes contain complex regions which are difficult to sequence with standard protocols, the lab must use a multitude of techniques specialized for each project. Problematic regions, for example, can be GC-rich or contain hairpin loops, have long homopolymer stretches, can be AT-rich, and/or contain tandem repeats of variable length. Gap closer in such regions is expensive as well as time-consuming, since it requires extensive troubleshooting strategies. Approaches include, optimizing reaction conditions, applying various sequencing chemistries, sequencing the opposite strand, and additional manual editing. For genomes with ? 65% GC content, we use a four step approach to sequence through difficult regions: DMSO, Sequence Finishing Kit (SFK), PCR, and shatter libraries. This strategy has allowed JGI ? s Microbial Genome Finishing Group to complete a number of complex microbial projects, such as, Frankia (~75% GC-rich) and Thermobifida fusca (~68% GC rich).