Sequencing Surge

How two new methods and two years have changed sequencing.

Edyta Zielinska
Oct 1, 2007
<figcaption>Roman Thomas from the Dana-Farber Cancer Institute and colleagues used 454 Life Sciences' sequencing to search for low-frequency mutation in a mixed-tissue lung cancer sample. At left, the sequencer detected a point mutation at a relative allele frequency of about two percent. Credit: Source: Nature Medicine</figcaption>
Roman Thomas from the Dana-Farber Cancer Institute and colleagues used 454 Life Sciences' sequencing to search for low-frequency mutation in a mixed-tissue lung cancer sample. At left, the sequencer detected a point mutation at a relative allele frequency of about two percent. Credit: Source: Nature Medicine

In the past two years, methods to sequence DNA faster and cheaper gave scientists the ability to "bring sequencing back into the lab," says Neil Hall, professor of Genomics at the University of Liverpool, and to ask genome-wide questions that would have been too expensive and time consuming using traditional Sanger sequencing at one of the large sequencing centers worldwide.

Each of the new sequencing methods, one marketed by 454 Life Sciences (published in a 2005 Hot Paper that has been cited 246 times, Nature 437:376-81, 2005) and another by Illumina, has preferred applications, says Elaine Mardis, codirector of the Genome Sequencing Center at...

Epigenetics

This May researchers published the first peer-reviewed research using the Solexa sequencing instrument. They profiled nucleosomal DNA to search for histone-methylation in order catalogue all methylations across the entire genome. Keji Zhao at the National Heart, Lung, and Blood Institute and the principle investigator of the study says that the Solexa instrument could sequence all the DNA they derived in a single run. "The 454 would take 10 times more sequencing runs" at a prohibitively higher cost, says Zhao.

A. Barski et al., "High-resolution profiling of histone methylations in the human genome," Cell, 129:823-37, 2007. (Cited in 3 papers)

Metagenomics and the microbiome

Researchers used 454 sequencing to study the effects of different microbial communities in the gut of obese mice. The sequencing doesn't use bacteria to amplify DNA, thus overcoming the "cloning bias" caused by using a live organism that does not replicate toxic genes or that contains stop codons. The authors could "sample the entirety of microbes that are present in the sample," says Mardis, who was a coauthor on the study. The study found that the microbiota of the obese mice has a different makeup compared to lean mice, making more energy available to the host.

P.J. Turnbaugh et al., "An obesity-associated gut microbiome with increased capacity for energy harvest," Nature, 444:1027-31, 2006. (Cited in 17 papers)

Cancer genetics

Finding the specific genes that are involved in human cancer has been complicated by the fact that a tumor is an amalgam of cells, some of which may be normal tissue. In collaboration with 454 Life Sciences, researchers avoided this problem by sequencing each cell as if it were a separate organism. Using "ultradeep" sequencing that requires oversampling of DNA 12,000 times (which would have been too expensive using Sanger sequencing), researchers detected small mutations in oncogenes that affected the cell's susceptibility to anticancer drugs.

R.K. Thomas et al., "Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing," Nat Med, 12:852-5, 2006. (Cited in 11 papers)

Small RNAs

David Bartel at MIT's Whitehead Institute for Biomedical Research and colleagues have been using new sequencing technologies to investigate new classes of small RNAs. With standard sequencing in 2003, Bartel says he was happy to get 4,000 RNAs sequenced. In 2006, using 454 sequencing he could get 400,000, and this year, using the Solexa instrument, he'll get 50 million. Using the 454, Bartel and his colleagues recently found a new class of RNAs in nematodes, named 21U-RNAs for their consistent 21-nucleotide length that always begins with a uridine residue.

J.G. Ruby, "Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C. elegans," Cell, 127:1193-207, 2006. (Cited in 11 papers)

Want to sequence Drosophila? Here's what it takes for 100 Mb:
Oversampling needed Total cost (US) Time to run sequencer (1X)*
ABI 3730 - capillary/Sanger 8X 650,000 1.34 Mb in 24 hrs
454 Life Sciences 25X 132,000 100 Mb in 8 hrs
Solexa 25X 12,500 1 Gb in 3-4 days
*Source: Elaine Mardis, Washington University Genome Sequencing Center