Single Molecule Sequencing
Patrice Milos, Ph.D., vp and CSO at Helicos Biosciences, described the company’s genetic analysis system at Cambridge Healthtech’s “Next Generation Sequencing” conference in San Diego last month in a presentation entitled, “Enabling True Biology with Helicos™ Single Molecule Sequencing.” DNA sample preparation on the HeliScope™ Single Molecule Sequencer requires no ligation steps or PCR amplification, Dr. Milos said.
The basic process for quantitative RNA analysis involves creating a single-stranded copy of the total or poly A+ RNA sample using reverse transcriptase, adding a polyA tail to the cDNA, and allowing these to hybridize to a flow cell surface containing oligo dT to initiate sequencing by synthesis reactions of the single-copy transcript tags. The result is both sequence information and read counts for the transcripts present in the sample.
By eliminating the need for PCR, the Helicos technology offers “a truly unbiased view of biology,” said Dr. Milos. As an example she described transcriptome analysis of a human placental sample: “You will get well over 14,000 genes reported that range from very rare—1 to 2 transcripts/million—up to 100,000 transcripts/million. The 50-channel HeliScope can generate 12 to 14 million reads in a single channel, with a resolution in the range of one transcript/million.”
Dr. Milos reported that customers are beginning to use the Helicos system for a broad range of applications that combine accurate quantitation and sequence information, such as copy number variation (CNV) analysis, gene-expression profiling of different tumor types or cell lines, and discovery of novel transcripts.
Whereas, the current HeliScope can accommodate in the range of 12 to 14 million sequence tags per channel, Dr. Milos expects to see a rapid increase in throughput by about 2½-fold in the near future. Average read lengths, now 34–35 bases, will also increase.
“We are now enabling paired reads on the Heliscope,” she said. “This improvement allows the instrument to read two regions of a molecule in a single run, with the same sample preparation and will make it possible for customers to do more exploration at the whole genome level.”
A team of Swedish researchers from Uppsala University recently presented a random array format and associated decoding scheme for “targeted multiplex digital molecular analyses” in Nucleic Acid Research. This method analyzes DNA samples using sets of padlock or selector probes that identify target sequences and create circular DNA molecules, which are then amplified via rolling circle amplification.
Padlock probes are “linear oligonucleotides that become circularized in a strictly target-dependent ligation reaction.” Selector probes are similar to padlock probes, with “target-specific ends for target recognition, flanking a DNA sequence with elements for amplification,” but they differ in that they “are designed to hybridize to the end-sequences of restriction digested genomic DNA fragments and thus template DNA ligase assisted circularization of specific genomic DNA sequences.”