Profiling Populations and Diversity of Gene Pools with Highly Accurate Single Molecule Reads

Sequencing to profile complex populations of genomes or gene pools with similar sequences if often challenging with current sequencing technologies, if read lengths are shorter than the distance between sequence differences. Technologies for generating longer sequence reads have had limited use due to lower throughput, higher cost and lower accuracy. There have been recent improvements in all these areas with the introduction of the PacBio Sequel System, which can generate hundreds of thousands of very high accuracy, long single-molecule reads per SMRT Cell.

We have explored the Sequel System for profiling complex metagenomics communities, multiplexing large gene pools or diverse genes, and tracking sequence variation in viral populations, situations where short reads alone do not provide adequate information. Sequence targets range in size from 600 to several thousand bases. To maximize the number of passes on single-molecule templates, long movies were collected. The consensus reads were then filtered at predicted single-molecule accuracy levels up to 99.999%. The resulting data illustrate currently obtainable levels of accuracy by template lengths, and show the trade-offs between template length, accuracy, and yield.

We show data from a variety of complex population applications that span these capabilities, including a bovine rumen microbiome study, which can help guide decision-making and planning for projects involving sequencing of amplicons or WGS libraries in the 500 – 5000 base pair size range.