High-throughput whole genome resequencing on the Helicos® Genetic Analysis System provides accuracy and virtually unbiased coverage at low cost, providing the most cost-effective path to analyzing whole genomes. The System’s simple sample preparation provides an unbiased look at native genomic DNA from a sample while the high throughput enables the interrogation of multiple samples simultaneously at greater sampling depth.
The Advantages
Features of the Helicos Genetic Analysis System enable the most effective whole genome resequencing in the industry.
- Direct sequencing of individual DNA molecules makes amplification unnecessary
- Simple, amplification-free sample preparation eliminates biases related to ligation, as well as PCR-based copying processes
- Results demonstrate absence of sequence context bias and fragment size bias
- Delivers sequencing data from start to finish at costs similar to what others charge for only sample preparation
- Enables human genome resequencing using our unique paired-read approach
The Evidence: A Case Study of Bacterial Genomes
Helicos has documented success in whole genome resequencing. Using Helicos True Single Molecule Sequencing (tSMS)™ technology, Helicos scientists resequenced three bacterial genomes — Escherichia coli strain K12 MG 1655, Staphylococcus aureus strain USA 300 and Rhodobacter sphaeroides 2.4.1. The three species are reference genomes with highly divergent levels of GC content. The resulting data demonstrate the ability of the HeliScope™ Single Molecule Sequencer to achieve even and near-complete coverage across full genomes regardless of GC content. For access to data sets, visit the HeliSphere™ Technology Center, an open access Web site for Helicos data and bioinformatics tools.
| Species | Genome Size (Mb) | GC content (%) |
| E. coli K12 MG 1655 | 4.6 | 50.8 |
| S. aureus USA 300 | 2.8 | 32.7 |
| R. sphaeroides 2.4.1 | 4.3 | 68.8 |
Among the results of the project:
- As many as 10 million alignable sequence reads per channel, with consensus accuracies greater than 99.995% at 20X coverage
- One channel on a 50-channel flow cell was sufficient for obtaining >50X coverage of a whole bacterial genome – demonstrating the ability to sequence larger genomes or up to 50 different bacterial samples in a single 8-day run
- Significant GC or other spatial context biases associated with coverage or consensus accuracy are not observed
- Unbiased resequencing of bacterial reference genomes demonstrates the effectiveness of tSMS for quantitative applications such as copy number variation, digital gene expression and chromatin immunoprecipitation sequencing.
Figure 1. Absence of G+C content bias. The G+C content of the three bacterial genomes (Top panel: E. coli; Middle panel: S. aureus; Bottom panel: R. sphaeroides) is plotted by tabulating the frequency of 200 bp windows with given fraction G+C (black line, left axis). Unlike for amplification-based sequencing technologies, the average coverage obtained by Helicos™ True Single Molecule Sequencing is not affected by the G+C content, regardless of the genome analyzed (red line; right axis).
Figure 2. Histogram indicates depth of coverage of uniquely alignable reads across Rhodobacter genome. Histograms are similar for other species. Fraction with zero coverage represents repetitive elements in the genome, thus non-unique reads.
The Approach
Native genomic DNA from a sample is sheared, treated with terminal transferase to generate a poly-A tail and loaded onto the instrument. No ligation or PCR amplification steps are required. The tailed fragments hybridize to complementary poly-T strands anchored to the flow cell surface. Inside the HeliScope™ Single Molecule Sequencer, a series of nucleotide addition and detection cycles determine the sequence of each fragment. Open source data analysis software aligns the hundreds of millions of reads to a reference sequence.
Figure 3. Helicos DNA Sample Preparation Methodology.
Helicos Whole Genome Resequencing – The simplest, most affordable and unbiased view of a genome.