At Mount Sinai, Precise Sizing and SMRT Sequencing Yield Unprecedented Read Length

At the Icahn Institute for Genomics and Multiscale Biology at Mount Sinai in New York City, scientists use automated DNA sizing together with long-read sequencing to analyze clinical samples, conduct routine surveillance on microbes, and more.

Technology development expert Robert Sebra, Ph.D., relies on Single Molecule, Real-Time (SMRT®) Sequencing from Pacific Biosciences with BluePippin™ automated DNA size selection from Sage Science. Together, these tools offer a powerful solution and industry-leading read lengths that allow Sebra and other researchers to resolve repeat elements and structural variants, rapidly close microbial genomes, and measure epigenetic marks.

Sebra, an assistant professor of genetic and genomic sciences, is no stranger to the SMRT Sequencing platform: he spent five years working at PacBio helping to develop that technology. Ultimately, his belief in the system led him to join the Icahn Institute, where he would get to use the PacBio® sequencer in the field. “There was a lot to be gained by taking the technology and applying it in a clinical setting,” says Sebra, who came to Mount Sinai in 2012. “I had experienced firsthand the value of long-read sequencing and wanted to apply it to human and infectious disease research.”

Since its founding by Eric Schadt in 2011, the Icahn Institute has attracted some 150 leading scientists and clinicians who bring a network-based approach to various biological questions, many of them focused on cancer, Alzheimer’s disease, allergy and asthma, and infectious disease. Among the institute’s well-stocked core facilities are two PacBio RS II sequencers and a BluePippin instrument, which are used together for projects requiring extra-long reads.

The PacBio RS II is his go-to system for epigenetic profiling, finishing microbial genomes, and exploring DNA samples likely to have repeats, large structural rearrangements, or ones that require allelic or accessory genome phasing.

As he applies long-read sequencing to these projects, Sebra continually looks for ways to generate the longest possible reads. One complementary technology for the PacBio workflow is BluePippin, an automated DNA size selection platform from Sage Science. Removing smaller fragments from the sequencing library ensures that the PacBio platform focuses on the longest fragments, so accurate sizing can improve average read length considerably. “You could do a traditional pulsed field gel every time you’re trying to size select, but it takes too much time, doesn’t scale well, and the DNA input requirement is really high,” Sebra says. “BluePippin is fast and cheap, and it’s the only option for size selecting in a high-throughput fashion. We purchased one as soon as it was available.”

Since bringing in BluePippin in 2012, Sebra’s team has run more than 100 libraries using the BluePippin+PacBio combo — in fact, he says, “For projects requiring near finished genome assembly, I don’t think we’ve prepared a library without BluePippin size select since owning the instrument.” He has been pleased with the amount of size-selected library the technology yields, noting that in virtually every experiment it produces more than enough to sequence a genome to completion on the PacBio RS II. He generally excludes all fragments smaller than 10 Kb to target the ultra long fragments, but says that in cases where input DNA is especially low or the genome is quite large and requires more library, he lowers that threshold to 7 Kb.

Sebra notes that the size selection step has exceeded his expectations for overall improvement in read length and throughput of SMRT Sequencing. The boost to mean read length from adding BluePippin size selection ranges from about 30 percent to 125 percent, depending on the input quality, he says.

In one infectious disease study, the team sequenced multiple MRSA isolates using PacBio with and without BluePippin sizing, finding that prior to sizing, 50 percent of the bases are in reads 5 Kb or longer, while after sizing that number more than doubled to 12.5 Kb.

“If your throughput of [PacBio] runs is high enough, a BluePippin is really pretty affordable,” Sebra says. “Size selection reduces the number of SMRT Cells required to achieve a particular sequencing goal, so it pays for itself pretty quickly.”

For more about Sebra’s scientific and clinical efforts, check out the full case study here.

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