AllSeq got a sneak peek at the latest sequencing library construction technology from iGenomX yesterday. The company, which was spun out of The Scripps Research Institute in San Diego, CA, is based on the work out of Dr. Daniel Salomon’s lab, where he continues to lead the R&D efforts. By eliminating most enzymatic and handling steps, the iGenomX prep promises to simplify and speed up the library construction process. More importantly, they feel their prep offers much more uniform coverage of the genome, especially when paired with long range, linked-read platforms.
How does it work?
The iGenomX technology aims to eliminate sequencing errors that plague other library prep methods by replacing the typical fragmentation and enzymatic manipulation steps with a simple random primer extension and termination.
- In the first step, P5 adapter tailed random 8-mers are hybridized to genomic DNA and then extended with polymerase. The fragments are terminated by the incorporation of biotinylated ddNTPs, and by tuning the dNTP/ddNTP ratio, the fragment length can be relatively controlled.
- In the second step, the fragments are isolated by binding to streptavidin beads. P7 adapter tailed random 8-mers are hybridized and extended via a displacing polymerase (ensuring that shorter sub-fragments are washed away).
- In the third and final step, the full-length fragments (with adapters at both ends) are PCR amplified and library products are size selected and ready for sequencing.
Why is it important?
While this method could be applied to a number of sequencing applications, the team at iGenomX is first focusing on improving genomic coverage with micro-droplet based linked reads. While their method will be platform agnostic, much of the early work has been performed on a RainDance machine. In early runs of their technology, runs are achieving coverage where 90% of the bases are within one standard deviation of the mean. In other words, if they’re hitting an average coverage of 30X across all the bases, the vast majority of the bases are pretty close to 30X (with many fewer bases at very low or very high coverage, which is typical for most sequencing datasets). And while they aren’t talking about it too much right now, they’re adapting the technology for a stranded RNA-Seq protocol with extension and amplification directly from RNA with no intermediate cDNA steps.
When can you get it?
While the product hasn’t launched yet, interested parties should be able to get their hands on some iGenomX data soon. Within the next couple of months they intend to place some data from the “Genome in a Bottle” sample NA12878. They’re aiming for a full commercial launch at ASHG in October, with alpha and beta testing between now and then.