from Genetic Engineering News- Third-Generation Sequencing Debuts
by Vicki Glaser
In Helicos’ tSMS technology, labeled nucleotides are mixed with nucleic-acid templates immobilized on a flow cell. Detection of the fluorescent signals emitted as a result of each base addition is performed in the HeliScope™ Genetic Analysis System. The Helicos system can sequence 105–180 megabases/hour with average read lengths of 33–36 bases from templates ranging in length from 25 to 5,000 bases.
It will enable a range of applications including chromatin profiling by direct sequencing of immunoprecipitated DNA, direct RNA sequencing, small RNA quantitation, digital gene expression, copy number variation assessment, and epigenetic analysis.
For direct RNA sequencing, the system can produce 300–400 million aligned reads/run with an average read length of 34 nucleotides (range 25–55) and a <5% per nucleotide error rate. Dr. Milos presented qualitative and quantitative data from RNA studies using tSMS to map the 3´ ends of RNA transcripts from yeast and human liver cells, producing a high-resolution map of 3´ polyadenylation sites. Another project under way is using direct RNA sequencing to study a pool of micro-RNAs and generate miRNA count distribution. Early results suggest that the technique yields greater quantitative accuracy than conventional cDNA-based methods.
In Pacific BioSciences SMRT™ sequencing technology, sequencing takes place on SMRT cells, each of which contains thousands of zero-mode waveguides (ZMWs). Each ZMW represents a hole tens of nanometers in diameter in a metal film that has been deposited on a silicon dioxide substrate.
The PacBio system generates both DNA sequence and epigenomic information directly from the real-time sequencing of genomic DNA. Single-molecule sensitivity enables faster results and longer read lengths.
Within about two years, the company plans to offer an application that will enable direct RNA sequencing in real time on the SMRT system without the need to convert RNA to cDNA. This application will provide insights into the epigenetics of RNA. An example was presented at the AGBT conference in which RNA sequencing using SMRT technology could distinguish pseudo-uridine from its native analog.