Early-access customers of a new sample loading device that Pacific Biosciences launched last week say that the upgrade improves results for certain samples, reduces contamination, and cuts down the amount of library material required per run.
PacBio released the MagBead station and reagent kit as an upgrade for its sequencer, the PacBio RS, last week. The device, which every customer will receive free of charge, uses magnetic beads coated with library material and helps to load the SMRT cell wells, or zero-mode waveguides, more efficiently with DNA and polymerase. According to the company, it also increases the percentage of long reads, cuts down on sample usage, and makes sample prep for the PacBio more robust overall.
"We expect that our automated MagBead station will make a substantial difference in customers' ability to run experiments on challenging samples because it will reduce the impact of the variability in sample prep while reducing the amount of sample needed to perform highly accurate sequencing," said PacBio CEO Michael Hunkapiller in a statement.
At Expression Analysis, which has had access to the MagBead upgrade for a little more than a month, one of the main benefits, though unanticipated, has been that a number of difficult DNA samples that had previously been "very recalcitrant" to sequencing on the PacBio now yield sequence data.
These samples "simply did not want to sequence; they wouldn't load into the ZMWs properly," Patrick Hurban, the company's vice president of research and development, told In Sequence. The samples did not have anything obvious in common, he said, and included plant, bacterial, and fungal DNA. "For every case that we have looked at so far, the MagBead station has allowed us to recover sequence," he said, adding that as a result, "we're much more confident on a sample-by-sample basis that we will be able to get good sequence."
In addition, the amount of material from a standard library prep that needs to be loaded is now about six to eight times smaller than before, he said. Although Expression Analysis has not tested this yet, this probably means that DNA libraries can be made with less DNA, and Hurban said that PacBio has already published a protocol that starts with less material.
For small bacterial genome sequencing projects, EA typically starts with 8 micrograms of DNA today, he said, and this could potentially be reduced to as little as 1 microgram with the MagBead.
This, in turn, could restart some project discussions with clients who were interested in using the PacBio but were unable to provide several micrograms of DNA.
The MagBead procedure also seems to favor larger DNA fragments over smaller ones – potentially because smaller DNA fragments coupled to a magnetic bead cannot reach the bottom of the ZMWs – which increases the percentage of longer reads in a run. As a result, small contaminating DNA fragments, including adapter dimers, are excluded from the SMRT cells. "So even with libraries where we had visible contamination with smaller sequences … we have now virtually eliminated these small sequences with MagBead," Hurban said.
Libraries with large insert sizes in particular − for example, 5 to 6 kilobases, or 8 to 12 kilobases – seem to work well with the MagBead station, and Expression Analysis is about to test whether this is also true for smaller insert sizes. "The main benefit of the MagBead upgrade is that we are now able to see the same kind of loading dynamics with very large insert libraries that we always had with smaller insert libraries," Hurban said.
Before the upgrade, about 30 percent to 45 percent of ZMWs were typically active after loading, depending on the type of DNA library, and now it is typically between 50 percent and 60 percent, he said.
It is still possible to use the PacBio RS without the MagBead station, which can be chosen through the instrument's control software, and the device adds only about 30 minutes of extra time to a typical run of 16 SMRT cells, which take a day and a half to load and run, he said.
Other users appear to have had similar experiences. "The MagBead station greatly reduces any adapter-dimer that may be present in some libraries and there are clearly benefits in terms of how much library is consumed during loading a sequencing run," said Chad Nusbaum, scientific director of Broad Technology Labs at the Broad Institute, in a statement from PacBio. "Compared to diffusion loading, approximately one-tenth of the library is needed to load each SMRT cell, allowing us to contemplate much deeper sequence coverage from a single library preparation," he said.