Pacific Biosciences Single Molecule, Real-Time (SMRT) Sequencing technology provides the most comprehensive view of genomes, transcriptomes, and epigenomes.
Image: Courtesy of Pacific Biosciences of California, Inc.
SMRT sequencing technology generates reads tens of kilobases in length allowing to assemble complete genomes and sequence full-length transcripts. Technology is free of systematic errors and achieves >99.999% consensus accuracy. No bias based on GC content means sequencing through regions inaccessible to other technologies. Capturing sequence data from native DNA or RNA molecules enables highly accurate long reads with >99% single-molecule accuracy. With no PCR amplification step, base modifications are directly detected during sequencing.
To learn more about technology, visit Pacific Biosciences site.
NGI offers de novo sequencing using Sequel II system
Reference genomes are currently commonly assembled de novo using only long-read technologies because it results in long contiguous assemblies (contig N50 over 1 Mb) with a few scaffolds.
For a good reference-quality assembly, one should aim for 20x (minimum 15x) of effective coverage per allele with HiFi reads. NGI currently recommends PacBio for production of the bulk sequencing data. For data consisting of a high number of PacBio HiFi reads, no additional polishing with Illumina is required, except for HiC/OmniC for scaffolding.
For specific DNA quality requirements for long-read sequencing technologies see the document (link to DNA quality document).
At present (2021.01.31), NGI requires 15 ug of chemically pure HMW-DNA (estimated by Qubit) for HiFi libraries that need to be sequenced on 1-3 SMRT cells. The higher the number of cells required, the more DNA one has to provide.
A 20 kb library can be prepared from as little as 150 ng input material, however, this option is only suitable for genomes under 300 Mb.
There are options of using 3-5 ug DNA for constructing long genomic libraries as well, but those might not be applicable to all of the projects. Please contact NGI for more details.