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BWH-Led Team Uses 454 GS 20 to Sequence Lung Cancer Transcriptomes

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A public-private research team led by doctors at Brigham and Women’s Hospital in Boston has used 454’s GS 20 platform to sequence and analyze the transcriptomes of lung tumors, according to a recent study.
 
The team, which included scientists from 454 Life Sciences, the National Center for Genome Resources, and a company called RxGen, sequenced cDNA from four samples of malignant pleural mesothelioma, or MPM, and two controls.
 
Unlike other cancer sequencing projects that have focused on genomic DNA to discover mutations, this approach simultaneously provided information both about gene expression levels and mutations in the expressed genes.
 
“I think this is a very powerful approach that will be used more and more widely in the future,” Matthew Meyerson, a researcher at Dana-Farber Cancer Institute, told In Sequence in an e-mail.
 
Study leader David Sugarbaker, chief of the division of thoracic surgery at Brigham and Women’s Hospital and a professor at Harvard Medical School, added that the research “is really an attempt at an unbiased complete sequencing of a transcriptome of an individual patient’s tumor.”
 
The goal of the study, which appears online in the Proceedings of the National Academy of Sciences this week, was to discover mutational profiles in MPM. The hope is to find mutations that sensitize certain tumors to specific drugs, and to use these mutations to select treatments.
 
“Ultimately, we view this as a future diagnostic tool,” said Raphael Bueno, associate chief of thoracic surgery at Brigham and Women’s Hospital, and the senior author of the study.
 
Unlike microarrays, which only reveal expression levels of genes, sequencing the transcriptome could also find mutations in these genes as well as uncover genes that might not appear on an array.
 
‘Technically Easier’
 
Almost two years ago, Meyerson’s laboratory published a study in Nature Medicine that used 454’s sequencing platform to look for mutations in the epidermal growth factor-receptor gene in the DNA of lung adenoma samples (see In Sequence’s sister publication, GenomeWeb Daily News 7/24/2006).
 
It was around that time that the BWH-led team began sequencing MPM, which is linked to asbestos exposure. Back then, 454’s technology was the only next-generation sequencing technology available, and the GS 20’s relatively long reads of approximately 100 bases made the project “technically easier” than the ultra-short reads that other platforms provide, and had the potential to find splice variants, said Bueno.
 
The reason the project took so long is that researchers had to overcome a number of hurdles to establish a pipeline that starts with a tumor biopsy and ends with the transcriptome sequence.
 
The first challenge was to come up with a tissue sample-preparation protocol that could provide high-quality tumor RNA and contain few normal cells or necrotic tissue. To that end, the researchers developed a microaliquoting technique in which they slice a tumor and examine every other sliver under a microscope. The intervening slices, based on their tumor content, are used for RNA preparation.
 

“Future cancer genome efforts will have to complement whole-genome sequencing with expression analyses of the observed mutant alleles.”

For the data analysis, the researchers teamed up with the National Center for Genome Resources in Santa Fe, NM, which used its Alpheus software to map the sequence reads to approximately 19,000 well-curated mRNAs in the RefSeq database, call variants, and display the data and results online. The data can be viewed here.
 
In total, the scientists found 15 novel mutations of different types in the four MPM samples, including seven point mutations and three deletions. Interestingly, each of the tumors had a unique mutational profile.
 
The cost of sequencing a tumor sample’s transcriptome is on the order of tens of thousands of dollars and takes six to eight weeks, according to the researchers.
 
While other studies have used methods such as SAGE for sequence-based gene expression profiling, the PNAS paper “represents one of the first studies to specifically investigate the feasibility of large-scale sequencing to simultaneously generate gene expression profiles and detect somatic and germline variants by shotgun sequencing of tumor cDNA,” Levi Garraway, an assistant professor of medicine at Harvard Medical School and at the Dana-Farber Cancer Institute, told In Sequence by e-mail.
 
However, he said that a disadvantage of transcriptome shotgun sequencing is the “uneven and incomplete coverage” that tends to “oversample the hyperabundant transcripts while achieving incomplete coverage of a large fraction of expressed genes.”
 
Meyerson agreed that the drawbacks of the approach are a “lack of representation of genes with low expression levels” and “the high cost of the assays.”
 
In their article, the scientists said they observed around 15,000 RefSeq genes in each patient sample, but many of these were represented by only a few reads.
 
According to Garraway, the technique could be improved by cDNA normalization and by using paired-end reads that would be able to identify splice variants or new gene fusions.
 
However, a potential advantage of the approach is that it could pick up mutations in genes that are expressed but not annotated yet, said Tobias Sjöblom, a researcher at Uppsala University in Sweden. On the other hand, some mutations that decrease the stability of mRNA might not be picked up by the approach. 
 
“The sensitivity of the technology for somatic mutation analyses would be easier to assess if the sample size was larger and some tumors had known mutations, in a gene such as TP53,” serving as a positive control, he said.
 
The study shows that “future cancer genome efforts will have to complement whole-genome sequencing with expression analyses of the observed mutant alleles,” according to Sjöblom. In 2006, as a researcher at Johns Hopkins University, Sjöblom and colleagues published a study in which they sequenced 13,000 genes from the DNA of 22 breast and colorectal tumors by Sanger sequencing (see GenomeWeb Daily News 9/12/2006).
 
Currently, the BWH-led team is sequencing an unspecified number of additional MPM samples in collaboration with 454, and has already been “finding some very interesting things,” according to Sugarbaker.
 
Sugarbaker, who is also the founder and director of the International Mesothelioma Program, which aims to understand the causes of MPM and develop new therapies, said the plan “is to scale this up and to develop a larger understanding of the profile in mesothelioma patients, and then to extend that to other solid tumors.”
 
“By sequencing dozens to several hundred tumors, we will be able to narrow down the vast majority of the tumors and get better clues on the pathways and targets for therapy,” Bueno said.
 
But he is not sure if the researchers will always use 454’s technology. “The nice thing about this is that the software can work for anything,” according to Bueno. “So whatever the best technology is, we can use it. Right now, we feel that 454’s technology is best suited. But if someone comes out with better technology, [it] can work, too.”

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