New Sequencing Technology Reveals Thousands of Unknown Genome Variations

 :: Posted by American Biotechnologist on 11-10-2014

Thousands of never-before-seen genetic variants in the human genome have been uncovered using a new genome sequencing technology. These discoveries close many human genome mapping gaps that have long resisted sequencing.

The technique, called single-molecule, real-time DNA sequencing (SMRT), may now make it possible for researchers to identify potential genetic mutations behind many conditions whose genetic causes have long eluded scientists, said Evan Eichler, professor of genome sciences at the University of Washington, who led the team that conducted the study.

“We now have access to a whole new realm of genetic variation that was opaque to us before,” Eichler said.

Eichler and his colleague report their findings Nov. 10 in the journal Nature.

To date, scientists have been able to identify the genetic causes of only about half of inherited conditions. This puzzle has been called the “missing heritability problem.” One reason for this problem may be that standard genome sequencing technologies cannot map many parts of the genome precisely. These approaches map genomes by aligning hundreds of millions of small, overlapping snippets of DNA, typically about 100 bases long, and then analyzing their DNA sequences to construct a map of the genome.

This approach has successfully pinpointed millions of small variations in the human genome. These variations arise from substitution of a single nucleotide base, called a single-nucleotide polymorphisms or SNP. The standard approach also made it possible to identify very large variations, typically involving segments of DNA that are 5,000 bases long or longer. But for technical reasons, scientists had previously not been able to reliably detect variations whose lengths are in between — those ranging from about 50 to 5,000 bases in length.

The SMRT technology used in the new study makes it possible to sequence and read DNA segments longer than 5,000 bases, far longer than standard gene sequencing technology.

This “long-read” technique, developed by Pacific Biosciences of California, Inc. of Menlo Park, Calif., allowed the researchers to create a much higher resolution structural variation map of the genome than has previously been achieved. Mark Chaisson, a postdoctoral fellow in Eichler’s lab and lead author on the study, developed the method that made it possible to detect structural variants at the base pair resolution using this data.

To simplify their analysis, the researchers used the genome from a hydatidiform mole, an abnormal growth caused when a sperm fertilizes an egg that lacks the DNA from the mother. The fact that mole genome contains only one copy of each gene, instead of the two copies that exist in a normal cell. simplifies the search for genetic variation.

Using the new approach in the hydatidiform genome, the researchers were able to identify and sequence 26,079 segments that were different from a standard human reference genome used in genome research. Most of these variants, about 22,000, have never been reported before, Eichler said.

“These findings suggest that there is a lot of variation we are missing,” he said.

The technique also allowed Eichler and his colleagues to map some of the more than 160 segments of the genome, called euchromatic gaps, that have defied previous sequencing attempts. Their efforts closed 50 of the gaps and narrowed 40 others.

The gaps include some important sequences, Eichler said, including parts of genes and regulatory elements that help control gene expression. Some of the DNA segments within the gaps show signatures that are known to be toxic to Escherichia coli, the bacteria that is commonly used in some genome sequencing processes.

Eichler said, “It is likely that if a sequence of this DNA were put into an E. coli, the bacteria would delete the DNA.” This may explain why it could not be sequenced using standard approaches. He added that the gaps also carry complex sequences that are not well reproduced by standard sequencing technologies.

“The sequences vary extensively between people and are likely hotspots of genetic instability,” he explained.

For now, SMRT technology will remain a research tool because of its high cost, about $100,000 per genome.

Eichler predicted, “In five years there might be a long-read sequence technology that will allow clinical laboratories to sequence a patient’s chromosomes from tip to tip and say, ‘Yes, you have about three to four million SNPs and insertions deletions but you also have approximately 30,000-40,000 structural variants. Of these, a few structural variants and a few SNPs are the reason why you’re susceptible to this disease.’ Knowing all the variation is going to be a game changer.”

Thanks to University of Washington Health Sciences for contributing this story.

I’m a Grad Student!

 :: Posted by American Biotechnologist on 11-06-2014

Programming Cancer Cells to Self-Destruct

 :: Posted by American Biotechnologist on 11-05-2014

Automated Droplet Generator Saves Time and Improves Data Quality

 :: Posted by American Biotechnologist on 11-03-2014

Bio-Rad Laboratories announced the launch of its Automated Droplet Generator (AutoDG™ Instrument) for digital PCR. Combined with the QX200™ Droplet Reader and a laptop computer with QuantaSoft™ Software, the QX200™ AutoDG™ Droplet Digital™ PCR System provides a worry-free, automated way to generate droplets for high-quality data.

“With a quick setup, you’re able to walk away and return to a full plate of droplets ready for thermal cycling and analysis,” said Carolyn Reifsnyder, Bio-Rad marketing manager, Digital Biology Center.

Generating droplets with a hand pipet can be labor intensive and time consuming, and variations in pipetting habits among different users can cause some degree of inconsistency in the resulting data. Bio-Rad’s AutoDG Instrument creates droplets quickly, reproducibly, and reliably. At maximum capacity, the system can generate droplets for 96 wells in less than 45 minutes.

“Droplet Digital PCR is sensitive enough to detect differences in people’s pipetting habits,” said Viresh Patel, Bio-Rad senior marketing manager, Digital Biology Center. “This instrument eliminates that variability.”

The AutoDG Instrument has its own hood and HEPA filter, reducing contamination so researchers can create droplets on a standard laboratory bench, without the need for a PCR cabinet or cleanroom. Adding to the instrument’s ease of use is a color touch-screen interface and deck lighting that guides the operator through system setup to ensure no steps are forgotten. Confirmation is provided once the required consumables are loaded correctly and 45 minutes later the droplets are ready.

Bio-Rad’s AutoDG Instrument can generate droplets for fluorescent probe–based detection or EvaGreen dye–based detection. The instrument is compatible with the QX100™ and QX200 Droplet Digital PCR Systems.

For more information on Bio-Rad’s AutoDG Droplet Digital PCR System, please visit www.bio-rad.com/AutoDGpr.

Don’t Mess With Nature!

 :: Posted by American Biotechnologist on 10-30-2014