Conducting Massively Parallel Sequencing

One of the major breakthroughs in modern biology is the development of massively parallel sequencing, also called next generation sequencing (NGS), which enabled the complete delineation of the human genome more than a decade ago. Since then many more species’ genomes have been sequenced, and the cost per genome has dropped from billions to mere thousands of dollars. New discoveries are being made as a result of the capability many research teams now possess to not only sequence chromosomal DNA, but also to identify which regions a protein of interest specifically binds (Chip-seq), analyze a whole transcriptome of a cell population under investigation (RNA-seq), or find out which RNA regions an RNA binding protein resides (CLIC-seq).

While it is inevitable that many PIs will seriously consider the inclusion of deep sequencing in their next grant proposal, it is not necessarily easy to take the first step and get their feet wet, so to speak. Knowing what format (e.g. 454 for longer reads, HighSeq for higher accuracy, or Ion Torren for bench top convenience) to use and how much to pay requires a vast amount of knowledge and experience. Even when you are done with sample prep, amplification and sequencing, to handle such massive amount of data is not trivial—transporting data alone can be a headache. A database server for storage and analysis requires another layer of expertise. There is no easy solution but to get started somehow. However, be prepared to deal with these issues.

Whether the cost on a type of next generation service is justifiable depends on whether it is required for your purposes. For example, when analyzing a person’s propensity of developing a disease by using known, disease-relevant genetic information, often times exome sequencing is sufficient. This costs anywhere between $1,000 to $3,000 with 100X coverage, significantly less than sequencing a complete genome which typically costs ~$5,000 at ~20x coverage.

High coverage sequencing of maternal blood DNA has been developed into clinically approved prenatal diagnosis of trisomy in Down’s syndrome and other chromosomal abnormalities. Transcriptome analysis helped the understanding of how reprogramming works when iPSCs are. Looking forward, with more routine use of deep sequencing we can predict with much more certainty the “off-target” effects of RNAi or cellular toxicity of chromosomal modifications enabled by ZFN, TALEN, or CRISPR. As a matter of fact, we believe that transcriptome sequencing should be required after each RNAi event to prove a specific linkage between knockdown and functions; similarly, whole genome sequencing results need to be provided after making a site directed chromosomal change in the future for high level publications.

*This blog partially resulted from discussions between Jiwu Wang and his colleagues, who are NGS experts at UCSD’s Cellular and Molecular Medicine, Moore Cancer Center, and BGI Americas.

Tags: CRISPR, deep sequencing, exome sequencing, footprint free, genomic modification, iPSCs, Massively Parallel Sequencing, NSG, TALEN, transcriptome, whole genome sequencing, ZFN

Competition from the Marketplace to the Courtroom

The hottest subject in the biological research equipment field has to be whole genome sequencing; hence it is no surprise that companies execute mergers and acquisitions in order to position themselves to go after their competitors in an attempt to corner this valuable market.

A bit of the background history: Illumina was started a decade ago to build DNA chip arrays by people with experience at Affymetrix, when the latter was the first and absolute leader in the DNA chip field. For years, rather than providing DNA chips, Illumina was known for generating revenue by selling oligonucleotides at 20% of the prevailing market price, essentially starting the low end oligo market. Just three or four years ago, it was a front page promotion on Invitrogen’s website to sell Illumina’s oligos through a production/shipping alliance, a cooperation previously unheard of in our field for such low price, non-commodity products. This move quite probably contributed to the decisions made by the more dedicated oligo company, IDT, to acquire local oligo production houses and move to the West coast (Allele opted out of such an acquisition and later did one of its own by taking over Orbigen and since moved into the viral systems and antibody fields). At that point when whole genome sequencing technologies were becoming mature and marketable, Illumina had performed brilliantly in out competing the previously dominant chip supplier Affymetrix, acquired Solexa, and quickly moved into the whole genome sequencing with Genome Analyzer and Genome Analyzer II, a move Affi’s management probably regretted not making.

In the years roughly around 2005-2007, Applied Biosystems, Inc. (ABI) was developing its own genome analysis equipment, the SOLiD system. It surely had a solid base to build on from its strong leadership in providing sequencer and analyzers for many years. Earlier in the year Invitrogen and ABI merged to form Life Technologies, pitching Invitrogen (now LifeTech) and Illumina in a collision course in battle for dominance in genomic analysis. In September, LifeTech brought suit against Illumina for patent infringement; in October Illumina countered with suits of its own. While the fight in court may be long and only sprinkled with occasional fireworks, the competition in the market could be fierce and should ultimately decide on whose technology is superior and offered at better prices. From the technical presentation made by sales teams to us during on site seminars, Solexa’s science sounded better. I was sitting next to Jay Flatley, CEO of Illumina at a San Diego biotech CEO dinner, and heard him predicting that the technology would advance and in a few years, one could get their own genome sequenced for about a thousand dollars, ~10% of the current cost! That’s simply innovation and competition at work. But watch out, a new wave of sequencing technologies based on single molecule capture might make the Illumina and LifeTech courtroom argument a moot point in the market.

Tags: biotech, Genome Analyzer, Illumina, Life Technologies, oligo, SOLiD, whole genome sequencing