Feature: Next next generation sequencing

By Branwen Morgan

It took nearly two decades to go from the release of the first semi-automated genome sequencer in the mid-1980s to the launch of Roche’s flagship 454 FLX next generation sequencer in 2005. The 454 is now one of three major players in the next gen market whose impact on the world of genomics cannot be underestimated. Just five years later we are poised to embrace the next generation of sequencing technology.

Professor Craig Cary, Director of the Sequencing Unit at Waikato University in New Zealand, says every time you move to a new platform you have to learn how to use it and that takes time. “There are punctuated steps in the evolution of technology. We are moving towards a whole new level of sequencing technologies where there are multiple affordable platforms; but all have inherent strengths and weaknesses.”

This means these next gen sequencers, exemplified by Illumina’s Solexa Genome Analyzer and the Applied Biosystems SOLiD System, together with 454, are likely to continue to be adapted for myriad use rather than being superceded by the next next gen. Indeed, despite the rapid changes in the sequencing landscape, Cary believes even the first generation capillary-based Sanger sequencing will persist. “No technology will suit all questions,” he says. “So I don’t see capillary reads ever going away, because they are single reads where each well is distinguishable and, for de novo sequencing, it’s the fidelity of Sanger sequencing that sets the bar.”

The new wave of sequencers, sometimes called the third gen, are creating deal of excitement because they will likely enable scientists to reach the goal of the $US1000 human genome. But to do this, the cost of the sequencing chemistry needs to drop to around $US0.0000005/base, or two millionths of a dollar per base (at a 10x coverage). Currently, the cheapest next gen sequencers cost about $US0.000001/base. And while this is a gigantic improvement on 1985 prices when sequencing cost roughly $US10/base, we are still well short of the price target.

The third generation of sequencing technology sees single molecules of DNA being sequenced without the need for cloning or PCR amplification and the inherent biases these procedures introduce. There are generally two types of detection methods for single molecule sequencing: those that rely on fluorescence and CCD capture, and those that don’t. Instruments that use the first of these detection methods include the Helicos Heliscope, launched in 2008; Pacific Biosciences single molecule real time sequencing (SMRT) machines, which have been shipped to their first customers; and Life Technologies-VisiGen system, which relies on fluorescence resonance energy transfer (FRET), and Life Technologies expects the first instrument will be placed later this year.

“There’s a lot of buzz around the Pac Bio platform, with reads expected to average over 1000 bases long; reaching and exceeding the read length of Sanger sequencing,” says Cary who travels to great depths, figuratively and literally – he has been to the ocean floor – and as far afield as Antarctica, to discover new forms of microbial life.

However, like the second gen machines, these systems require expensive excitation lasers, fluorescent reagents and CCD cameras. Another, not insignificant, cost comes from the storage of the digital images created by extremely high-resolution CCDs that equate to terabytes of data, not to mention the bioinformatics systems required to process the vast amounts of data generated.

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The third generation

The two non-fluorescing technologies operate via quite different methods, with detection systems based on tiny changes in electrical current or pH, thus removing the most expensive components, and associated costs, of sequencing instruments. Hence, in terms of achieving the $US1000 genome, it is likely to be one of these that will reach the goal first.

Both nanopore sequencing, from Oxford Nanopore Technologies (ONT), and Ion Torrent, from Mass Genomics (which was just acquired by Life Technologies), are based on silicon chips. The ONT chip contains hundreds of wells each covered by a lipid bilayer that contains a nanopore – which is a hole around one nanometre in diameter – with each pore an individual electrical channel.

Sequencing is based on exonuclease cleavage of the single DNA strand and detection occurs when the cleaved nucleotide falls through the pore, transiently disrupting the current. The change in current amplitude is unique for each base (A,G,C,T and 5-Methylcytosine - the direct reading of which is unique to nanopore technology). ONT plan to commercialise their nanopore sequencing system by the end of 2010.

Ion Torrent, while also relying on advances in semi-conductor technology, sequences by monitoring DNA synthesis. Single types of nucleotides are sequentially flooded across the chip. Nucleotide incorporation into the new DNA strand results in the release of a H+ ion, which is detected by the pH sensitive dielectric layer. These breadbox–sized benchtop instruments cost about $US45,000 (disposable chips with 1.5 million wells are about $US500) and even come with an iPod, of all things, pre-installed with an application to monitor runs and cycles in real time. How 2010 is that? The Ion Torrent Personal Genome Machine (PGM) sequencer is just making its way into American research labs now.

Whether these single molecule sequencing systems live up to their potential is yet to be demonstrated. “True comparisons with current platforms will only be possible when there has been enough customer access via independent purchase,” says Sydney geneticist, Dr Vanessa Hayes, who has just left Australia to join the J. Craig Venter Institute in San Diego.

Mark Crowe of the Australian Genome Research Facility agrees that it’s too early to tell whether these third generation sequencers will truly provide a breakthrough. “If the technology works, it could completely turn things around for genome sequencing,” he says. “But there are a lot of big ‘ifs’ there.”

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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.