Until recently, enriching for certain regions of the genome has been virtually impossible. Repeat expansions, extreme GC regions, and other genomic elements are very difficult to target using traditional enrichment methods. That’s why our new “No-Amp” targeted sequencing application — a streamlined, amplification-free approach based on the CRISPR/Cas9 system — is a valuable addition to the SMRT Sequencing toolbox.
The method was demonstrated in a recent PLoS One publication, and a new webinar delves into technical details of the protocol. Hosted by our own Paul Kotturi and Jenny Ekholm, the presentation offers an overview of uses for which the No-Amp method is beneficial, real-world examples of its results, and advantages it holds compared to traditionally used PCR and Southern blot techniques.
Kotturi kicked off the presentation with a look at the general advantages of SMRT Sequencing, including long reads, high accuracy, single-molecule resolution, simultaneous epigenetic detection, and uniform coverage. He also noted some recent performance metrics from the new Sequel II System: more than half of data is in reads >190 kb , and each SMRT Cell 8M generates up to 160 Gb of sequence data. With the HiFi sequencing mode that makes use of circular consensus sequencing, the system can achieve Q30 accuracy with just eight passes around a molecule.
Next, Ekholm stepped in to focus on the No-Amp application. Generating a sequencing library using the No-Amp method is relatively straightforward, the first step is to block the 5’ and 3’ ends of the genomic DNA, followed by the CRISPR/Cas9 digestion. To enrich for the region of interest guide RNAs are designed flanking each end of the targeted region, making them available for sequencing adapter ligation after the Cas9 digestion. The sequencing library is then cleaned up before sequencing. The No-Amp method takes two days (with less than four hours of hands-on time) and is compatible with both the Sequel System and the Sequel II System.
Users of the No-Amp method can multiplex target regions, samples, or both to maximize sequencing efficiency and minimize cost. Typical target insert sizes range from 4 kb to 6 kb, though scientists have successfully extracted even longer fragments with this process, Ekholm noted. The expected yield is hundreds of Q20 reads per target and the on-target rate for the No-Amp method is 40-60%, which translates to enrichment factors of 10,000-100,000 fold.
Later in the webinar, Kotturi discussed elements needed for this protocol: high-purity, high molecular weight DNA; 5-10 µg of DNA per SMRT Cell but only 1-2 µug / sample when multiplexing 5-10 samples / run; guide RNAs; barcoded adapters, if multiplexing samples; and a No-Amp accessory kit with primers and buffers. He also presented information about cost. In a five-sample multiplex workflow, the cost (U.S. list price) comes to $220 per sample. When multiplexing increases to 10 samples, the per-sample cost drops to $130 per sample. When multiplexing multiple targets per sample, these costs drop even further per locus. At PacBio, we routinely run 4 targets per sample.
If your research would benefit from capturing and sequencing regions that are otherwise intractable, this webinar is well worth your time. It also includes valuable information about data analysis and visualization, specific examples of targeting disease-associated repeat expansion regions, and much more.