Targeted Resequencing surveys DNA for genetic variation or mutations in genomic regions of interest. The technique can help identify associated alleles or perhaps even causative mutations in disease pathways or candidate gene regions by analyzing a limited set of target regions in a large number of patient samples. Helicos’ Targeted Resequencing sample preparation protocol is compatible with any available targeted region capture strategy.
The Advantages
The Helicos® Genetic Analysis Platform allows targeted resequencing with a simplified and cost-effective sample preparation protocol, which is highly scalable and amenable to automation to maximize sample throughput.
The HeliScope Sequencer generates over eight million alignable direct sequence reads per flow cell channel, allowing analysis of target regions up to 10 megabases of sequence per channel at 20X coverage. With 50 channels per sequencing flow cell pair and the use of DNA barcoding, up to 250 samples can be analyzed per sequencing run.
And because of Helicos’ revolutionary sample prep process, the assay doesn’t introduce any biases, providing you with even coverage of your target regions.
The Evidence: Polymorphism Detection with Unprecedented Sample Throughput
Due to the high accuracy inherent in Helicos True Single Molecule Sequencing (tSMS)™ technology, Helicos Targeted Resequencing has demonstrated its ability to effectively identify SNPs in disease-associated regions. Combining this polymorphism detection ability with the simplicity and scalability of the Helicos Targeted Resequencing application will allow for cost-effective high-throughput follow up required after disease association studies.
The p53 gene of five different individuals with genotypes in the HapMap database was resequenced at 100% coverage using the Helicos Platform. The Helicos Genetic Analysis System was capable of:
- Identifying all the known SNPs in the HapMap samples (confirmed by independent Sanger sequencing; Table 1).
- Identifying new, undiscovered SNPs in the HapMap samples (confirmed by independent Sanger sequencing; Table 2).
- Identifying new SNPs in regions that were refractory to sequencing when attempted by a sequencing service provider using standard Sanger sequencing (Table 3).
SNPs found by Helicos Platform that are in agreement with HapMap data
| HapMap sample |
NA12239 |
NA12248 |
NA12802 |
NA12814 |
NA10857 |
| Homozygous reference |
17 |
18 |
19 |
20 |
19 |
| Heterozygous |
2 |
2 |
2 |
3 |
3 |
| Homozygous non-reference |
2 |
2 |
0 |
0 |
0 |
Table 1 shows SNP calling agreement among results obtained with the Helicos platform, those obtained with Sanger sequencing and the HapMap database. Homozygous reference data refers to SNPs in the various individual samples that correspond to the human reference sequence, while data labeled “Heterozygous” refer to samples in which one allele corresponds to the human reference sequence and one is unique. Homozygous non-reference data refer to those in which both alleles agree between Helicos and HapMap yet differ from the NCBI reference sequence used.
SNPs identified by Sanger and Helicos
HapMap sample
|
NA12239 |
NA12248 |
NA12802 |
NA12814 |
NA10857 |
| Heterozygous |
4 |
3 |
5 |
9 |
4 |
| Homozygous non-reference |
1 |
1 |
0 |
1 |
1 |
Table 2 shows correspondence between results obtained by Helicos and Sanger sequencing for SNPs that do not appear in the HapMap database.
SNPs identified by Helicos, but not by Sanger (due to coverage)
| HapMap sample |
NA12239 |
NA12248 |
NA12802
|
NA12814 |
NA10857 |
| Heterozygous |
2 |
3 |
3 |
2 |
2 |
| Homozygous non-reference |
1 |
1 |
1 |
0 |
0 |
Table 3 shows SNPs not contained in the HapMap database that were identified by Helicos sequencing, but not by Sanger sequencing.
The Approach
The Helicos Targeted Resequencing application achieves high throughput with true direct DNA measurement, which minimizes sample preparation burdens and facilitates scale-up.
Figure 2. Helicos DNA Sample Preparation Methodology.
DNA samples, amplicons, pooled amplicons or captured genomic fragments from your target regions of interest are simply sheared, tailed and blocked on the 3’ end – all without laborious cloning, ligation or gel purification steps. DNA targets are then hybridized to a flow cell and sequenced directly on the HeliScope™ Single Molecule Sequencer.
Helicos Targeted Resequencing - The throughput and accuracy to carry out large scale studies to find gene variants or mutations in your genes or region of interest.