Applied Biosystems and Dr. Alain Nicolas

A Partnership for the Advancement of Science

Since its creation in 1909, the Institut Curie in Paris has brought together scientists and physicians with a common goal: to defeat cancer. Marie Curie's legacy of the "Curie model" is defined by the seamless interface between cutting-edge basic research and innovative and quality healthcare.

Centre National de la Recherche Scientifique (CNRS) research scientist and leader of the Recombination and Genetic Instability group, Alain Nicolas is investigating yeast biology and genetics, and spearheading the Institut's implementation of the SOLiD™ System from Applied Biosystems for human genetic and cancer research. We recently talked with Alain about how the Institut is using the next-generation sequencing platform to study genome instability and how mutations are produced in living cells during growth and development.

What is the goal of the yeast research you are conducting?

It is clear that from the study of basic processes of DNA replication, recombination, and repair that mutations of living cells are the source of defects in cell biology function. This is the case in human cancer cells, where the genome is highly rearranged. The goal of our research is to determine the origin of the changes in the genome structure in cancer cells using a yeast model system that is easy to manipulate and allows us to study cell mutations in great detail.

In general, how is next-generation sequencing technology helping you in this work?

Next-generation sequencing gives us a new perspective in understanding the global activity of a cell at the genetic level, and provides the highly detailed information we need to correlate activity with function. In my laboratory, this means we can look for mutations across the entire yeast genome—which consists of about 12 million base pairs—in a single experiment.

Why did you choose the SOLiD™ System from Applied Biosystems over other platforms for next-generation sequencing?

We chose the SOLiD™ System for its accuracy and speed. We are looking for a rare mutation, so we need to be able to detect just one or two changes out of an entire yeast genome. The SOLiD™ Systems barcoding technology also gives us great depth of coverage in multiplexed samples in a single run, which allows us to study many different strains in a short time.

What implications do these early results have for your research going forward?

We can move forward with confidence using our yeast model system in routine experiments because the SOLiD™ System allows us to systematically and accurately resequence entire genomes in our strain in a single run. Before, we had to use PCR or Southern blot analysis techniques to verify the quality of our strain after each transformation to modify a single gene, which slowed our research and allowed us to study only a small region of the genome at a time.

With the high sequencing capacity of the SOLiD™ System, we can make sure that the strain we are using has the correct genetic information across the entire genome and that our analysis of a particular cell line will not be tainted by accidental mutations that were generated by the process. Plus, now that we can look at an entire genome in about a week, we can accelerate our understanding of the variations between many different strains.

You mentioned that you are using your yeast model system work as quality control for the SOLiD™ System. What other kinds of research will the Institut Curie be conducting on the platform?

Our use of the SOLiD™ System will be very diverse because we are an academic center with many different fields of investigation—including genetics, molecular biology, immunology and cell biology, and genotoxicology—and each laboratory is interested in a different type of information at the genome level. Many will rely on the SOLiD™ System for resequencing or sequencing of tumor cells that have a lot of genome rearrangement.

For example, we expect to use the platform for targeted resequencing that helps our colleagues look at mutations in specific collections of "candidate" genes that are important for particular cancers types. We will also conduct studies on gene expression that entail transcription profiles in different cell types. Several laboratories are interested in epigenetic modification, or other changes that affect chromatin, so we will support multiple yeast, mouse, and human research projects using the ChIP-Seq chromatin technique followed by deep sequencing. We will also use the SOLiD™ System for applications that involve small RNA contents of the cell and quantification and differentiation in different cell types.

Our job is to ensure that we have the capacity to handle all of these multi-disciplinary research projects on the SOLiD™ Platform, which is why we are using our own initial

Let's talk a little more about each of these application areas, starting with targeted resequencing, gene expression, and transcription.

We are particularly interested in targeted resequencing of candidate genes for different types of tumors. SOLiD™ System will give us the opportunity to look at mutations in hundreds of genes in a short time, and with the enrichment protocol, it will also be highly cost-effective.

Transcription and gene expression applications will allow us to look at the variation of gene expression in many different situations in order to analyze and understand cell function, differentiation, and development.

And epigenetics?

Epigenetics has become very important in biological research because it not only controls gene expression, but also how genetic information is transmitted from cell to cell. The high throughput we get with the SOLiD™ System will allow us to investigate epigenetics using a technique called Chromatin Immunoprecipitation and Sequencing, or ChIP-Seq. It allows us to do things like map protein binding in the entire genome or look at specific modification of histones that control gene expression in the entire genome. We can also use ChIP-Seq to map other components of chromosome function like transcription and regional replication on the global level—in a single chromosome or an entire genome.

What work are you doing in the area of microRNA?

Several laboratories at the Institut Curie are interested in microRNA expression and content. This is a relatively new field in biology, and a very important one for medical research because microRNAs are now known to play a central role in the differentiation of cells. We are using SOLiD™ Small RNA Expression Kit to quantify known microRNA and to discover and isolate new microRNA as they develop in different cell systems.

The kits give us the high accuracy in the reading of the genome information to discover new microRNAs that are short, 20 to 30 nucleotides. And they give us the very high coverage required to study the level of expression of microRNAs, which dictates their functions.

What about whole genome transcription?

In addition to microRNA studies, we are all interested in replacing microarrays, where we could probe only what we put on the slide, with whole genome transcription analysis. We have learned that the genome, whether yeast, mouse, or human, is more transcribed than we ever thought and therefore there is a transcript in the cell that holds important information about function. Using the SOLiD™ Next-Generation Platform for deep sequencing will give us more detail, resolution, and quantitative information about transcriptome and messenger RNA expression.

What do you see as the most challenging aspect of whole genome sequencing?

I've been doing genetic research for about 30 years, and I have seen many advances and revolutions in the science. But the idea that we would be able to sequence the entire genome of a single individual or a cell line was something the research community could hardly imagine five or ten years ago. As a geneticist studying DNA mutations, this new capability is extremely exciting but it also presents challenges.

The biggest challenge we face with sequencing an entire genome is ensuring that we have the bioinformatics in place to analyze the immense volume of complex information it generates in a very short time. For example, we estimate that there are between 200 and 500 polymorphic variations between cells—deletions, translocations, major re-arrangements—that might be the cause of a particular cancer. It takes an incredible amount of data analysis to understand what this means in terms of chromosome structure and function.

How have you prepared in terms of bioinformatics capabilities to handle the volume of next-generation data?

We anticipated and prepared for the high volume of data by establishing a dedicated next-generation bioinformatics platform, analytical team, and data pipeline that supports close communication between the biologists and the bioinformaticians. It's key that these communities be able to collaborate in order to analyze the information. We are also focusing on cross-training the biologists in our laboratory in bioinformatics because we want them to understand both sides of a sequencing project—the biology and the analysis.

Tell us about the new research building that will soon be home to the SOLiD™ System.

When we purchased the SOLiD™ System about six months ago, we wanted to get up and running quickly so we used the little space we could find in the institute. In the next week, we will be moving to a new building dedicated to research in bioinformatics and developmental biology. This will make it even easier for team members to work together to transform raw data into valuable information. It emphasizes how important next generation sequencing is for the institute—not only in the research area, but also in medical science and medical genetics.

We have 25 laboratories ready-to-go on the SOLiD™ Platform, and we will probably need to expand our facilities and purchase more equipment in the future. I think this is the direction the entire international research community is heading.

What excites you most about your work going forward?

We were the first laboratory to implement the next generation SOLiD™ Platform in France. When we embarked on our studies, I must admit I was a little worried about keeping up with rapid advances in the technology and the fast pace of data acquisition. Could we quickly set up and master the platform? Could we easily look at single nucleotide polymorphisms in short experiment runs? Could we conduct experiments in support of all of the different areas of research at the institute? And the answer after six months of our quality-control experiment is, "Yes, we can!"

We also belong to the Canceropole Ile-de-France, which is comprised of different institutions devoted to researching cancer, so we already have requests from our colleagues beyond the Institut Curie. I think this new platform will help us advance the research projects of many laboratories by allowing them to study tumor DNA in very high detail. That is exciting for the future of research, treatment, and hopefully a cure.