Applications
Prokaryote Whole Genome
Overview
The Genome Sequencer FLX System facilitates the completion of near-finished draft sequences of multiple whole prokaryote genomes on a single 10-hour instrument run. Assembly of long shotgun reads (400 base pairs) yields minimal contigs and the addition of multi-span paired end reads (>150 base tags from each end of a 3 Kb, 8 Kb or 20 Kb span) orders contigs into as few as one scaffold. For some microbial genomes, use only one 8 Kb span paired end library to assemble genomes into a single scaffold per chromosome, without the addition of shotgun reads. The sequencing depth achieved with 454 Sequencing systems ensures accurate characterization of microbial or bacterial diversity, sensitive detection of even rare mutations, and rapid discovery of disease causing agents.
Download flyer and view assembly data: De Novo Sequencing of Small Genomes
Publications:
- Bertelli et al. The waddlia genome: a window into chlamydial biology. (2010) PLoS One 5:e10890.
- Tripp et al. Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium. (2010) Nature 464:90-4.
- Gilmour et al. High-throughput genome sequencing of two Listeria monocytogenes clinical isolates during a large food outbreak. (2010) BMC Genomics 11:120.
- Tauch et al. Ultrafast pyrosequencing of Corynebacterium kroppenstedtii DSM44385 revealed insights into the physiology of a lipophilic corynebacterium that lacks mycolic acids. (2008) Journal of Biotechnology.
Click here to see all Prokaryote Whole Genome publications.
Data Analysis Tools:
GS De Novo Assembler Software, GS Reference Mapper Software
How it Works:
Shotgun Sequencing
For shotgun sequencing large-size genomic DNA samples are randomly fragmented into small 300- to 800-base-pair fragments via physical shearing. Addition of adapters to the generated fragments creates a library of DNA fragments which is immobilized on DNA capture beads and individually sequenced on a PicoTiterPlate device as shown in the workflow description. The generated sequences are then assembled into a number of unordered and unoriented contigs using the GS De Novo Assembler Software and a consensus sequence is generated.
Figure 1: Assembly of sequences into contigs using the GS De Novo Assembler Software
Paired-End Sequencing
After assembly of de novo shotgun sequencing reads into contigs the generated contigs are ordered and oriented using paired-end reads. These paired-end reads have two 100-mer DNA segments on each side (paired ends) that were originally located either 3 kb, 8 kb, or 20 kb apart in the sequence of interest. The GS De Novo Assembler Software enables subsequent mapping of the 20-mer fragments to the generated contigs and thus ordering and orienting of the contigs into scaffolds. This combined information provides a high-quality draft sequence of the genome.
Figure 2: Ordering and orienting of contigs via paired-end reads