Second generation sequencing redux
The third generation upstarts may be on their way, but that doesn’t mean second generation sequencers don’t have more yet to give. Improvements to the technology continue to be made on a number of fronts. They include an increase in the number of wells/reads per plate, superior base-calling algorithms and CCD detection rates and resolution (so the depth of sequencing required can be reduced for the same accuracy), and creation of scaled-down versions of instruments that, cost-wise, will put them in reach of the smaller research laboratories.
Second gen technology is also being applied to the study of numerous ‘omics, including transcriptomics, proteomics and metagenomics. The short reads, high throughput and coverage of second gen sequencers are used to explore gene expression across multiple samples, uncovering novel, aberrant and rare transcripts that would otherwise be undetected against a background of highly abundant transcripts.
Proteomes can also be studied through a technique that combines ribosome profiling and deep-sequencing. Studying transcripts alone can be misleading, as not all of them will be translated into proteins. Those that are being translated, have a region of around 30 nucleotides protected by a bound ribosome. Thus, sequencing of DNA libraries that correspond to all protected RNA fragments in a cell gives a time-dependent snapshot of protein production.
The study of metagenomes (genomes from heterogeneous microbial communities) has benefited enormously from advances in sequencing methodologies. Samples of intestinal flora and organisms from extreme environments, such as mine shafts or deep-ocean vents, can be directly sequenced avoiding the bias introduced by cultivation methods. And metagenome analysis of human gut bacteria are yielding fascinating insights into their potential role in disease.
Biomedical research efforts using new sequencing technologies are also shedding light on cancer genetics. The International Cancer Genome Consortium, launched in 2008, aims to detect and determine the effect of mutations in 50 of the most common types of cancer by analysing genetic changes across large numbers of patients.
Andrew Biankin, who co-leads Australia’s effort to map changes in the pancreatic genome, says they already have data from more than a dozen patients. “Next gen sequencing is particularly well-suited for studying cancer because of its massive throughput and its ability to detect all types of genomic aberrations, including large structural variants (such as translocations, rearrangements, insertions and deletions). Mapping the genomic landscape of cancer is substantially more complex than mapping normal genomic variations,” he says.
The Australian researchers use the SOLiD platform, but validate their data with 454 and Sanger sequencing. When complete, the mass of data will yield knowledge not only about the primary genetic changes but also about epigenetic changes and alterations to the transcriptome. The resulting catalogue of information will underpin new therapeutics and move us closer to the foreseeable future of targeted personalised medicines.
Given all the renewed interest in sequencing, perhaps we are not yet in the post-genomics era. Moreover, despite the considerable reduction in sequencing costs and the increase in throughput, there are still some gains to be made by improving sequencing accuracy and reducing the extent of the redundancy currently needed to reliably assemble contigs.
The time needed for the sequence assembly and analysis step, which often exceeds the run time, is currently the bottleneck that needs to be unblocked before any contender gets close to the ArchonX prize for Genomics, which offers $US10 million to the first outfit that can sequence 100 human genomes in 10 days with 98 per cent coverage and an accuracy of no more than one error in every 100,000 bases sequenced, and at a recurring cost of no more than $US10,000 per genome. That target is still a long way off, but with the developments in second and third generation sequencing technologies, it comes closer to being a reality every day.
This feature appeared in the July/August 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.