“There is a definite move to multiplexing on many more platforms,” explained Dr. Baldwin. “We offer services for microarray-based profiling of RNA, microRNA, DNA sequences and modifications, and proteins. Molecular diagnostics is still predominantly at a point where you only measure one thing at a time. Mostly, basic research has driven our demand for highly multiplexed assays. But some of that research is finally being translated into clinical applications that use the power of multiplexed analysis.”
Dr. Baldwin noted that there are now ways to barcode the samples. “That way you can multiplex many samples, as well as analytes, thus increasing the throughput. For molecular diagnostics—FDA has approved the first wave of multiplexed tests—there is no technical reason we can’t use multiplex technology, but we are still in the process of discovering what relevance tests have and how they inform clinical decision.”
In one application recently approved by the FDA, the Pathwork Diagnostics microarray tests expressed several thousand genes to determine the tissue of origin for a metastatic tumor. “This approach is familiar to genomics researchers—take a tissue sample, extract RNA, and put it on a microarray, match the gene-expression profile to reference sets, and find the pattern,” noted Dr. Baldwin.
“But pathologists can usually accomplish the same thing with histology and immunology tools. There is a big need for this technology, and we need to be doing more of this in the clinical setting, but it must offer clear advantages or enhance and expand current clinical practice.”
Many basic research projects are at the stage of consolidating gene-expression signatures from whole-genome data sets into focused multiplex panels. More formalized clinical trials will be the next step.
“A lot of people appreciate the power of multiplexing technology. Genotyping for functional and structural genomics and measurements of gene expression and activity for functional genomics, are all applications that have benefited. Now it’s time for us to turn to translational services,” remarked Dr. Baldwin. “And to do this well, you have to get the clinicians onboard early. Every test has to be justified, validated, and implemented with patient care in mind.”
At the CHI meeting, Dr. Baldwin observed that the rapid development of several genomics platforms for microRNA (miRNA) discovery and detection has created opportunities for translation to biomarker applications.
“Detection assays range from high-throughput single-target methods, to custom multiplex panels, to comprehensive microarrays, genomic tiling arrays, and next-generation sequencing,” he explained.
“Clearly, lots of people are working on microRNAs from basic mechanisms through biomarker utility to impact on disease development and therapies. This is what it’s all about.”
Immune System Sequencing
miRNAs are critical regulators of most major cellular pathways such as cellular differentiation, apoptosis, and metabolism. Although most studies rely on the detection of previously reported miRNAs, the most powerful approach to identify and quantify expression of new miRNAs remains direct sequencing.
To this extent, Francois Vigneault, Ph.D., Church laboratory, department of genetics, Harvard Medical School, has developed a procedure to facilitate miRNA capture via ligation by barcoding samples, thus allowing multiplexing and considerably minimizing the per-sample cost on next-generation sequencing platforms. Most projects Dr. Vigneault is working on are related to the multiplexing capability of next-generation sequencing instruments. He is focused on miRNA analysis, as well as a technology they call the Personal VDJ-ome for sequencing the complete immune repertoire of single individuals.
“Last year we optimized microRNA analysis on next-generation sequencers. The great thing is that there is less than 1,000 of these in human cells; it’s not like messenger RNA, which have a much greater dynamic range and longer sequences,” said Dr. Vigneault. “Sequencing microRNAs can be affordable; a typical cost per sample on next-generation instrument is around $1,200. By combining multiple samples per lanes on a single flow cell we can currently bring that cost to around $25 per sample for microRNAs experiments.”
Dr. Vigneault also noted that microarray technology can’t keep up with next-generation sequencing. “The average price for analyzing one microRNA sample on a microarray is $400 and is limited to what is on the chip. The problem we now face is that the new next-generation sequencers we are developing are just too good at multiplexing and would require up to 5,000+ samples per sequencing run to be worth it.”
Immunosequencing is another methodology that benefits from high-throughput technology development. “We are developing technologies that will allow the analysis of the immune system of a single individual in unprecedented detail and low cost,” he added.
“Antigens, bacteria, virus, or cancer cells are recognized by receptors on B and T cells. All the millions of unique receptors required to accomplish this task are produced by genetic recombination of the V, D, and J segments, hence the name VDJ-ome. We are currently sequencing all of these receptors in a single experiment from one individual to study the evolution of his immune repertoire in time.
“With one blood draw, we will be able to tell you everything that you have been exposed to in your life and, ultimately, produce new drugs and antibodies against virtually any antigens of current diseases and the ones to come. This is only possible by developing new multiplexing technologies of extreme throughput.”