May 2, 2024  |  Plant + animal biology

Long-read sequencing myths: debunked.
Part 4 — plant and animal

HiFi Facts blog header image with plants in background

The world is moving at a fast pace, isn’t it? Climate shifts, growing populations, and changing ecosystems are stacking up some serious challenges for species all over the planet, including our own. Researchers like you, working to figure out the complexities of plant and animal biology, are the key to protecting biodiversity and sustaining agriculture in this changing world.

With advances in long-read sequencing technology, we’ve got a sharper toolkit. Now, we can detangle complex polyploid genomes and sequence through challenging regions like never before. These insights are crucial for conservation biology, evolutionary research, and all those big questions we’re chasing to understand and look after our world.

This is part four of our six-part myth-busting series, where we dispel common myths and misconceptions about PacBio sequencing in plant and animal genomics applications. (New to the series? Check out Part 1 – HiFi sequencing, Part 2 – human genomics, and Part 3 – cancer genomics.)

Myth #1:

PacBio HiFi reads are shorter than nanopore reads and less accurate than short reads. I can get “good enough” genome assemblies using nanopore technology with short-read polishing.


This statement is inaccurate.

It might sound too good, but it’s true—PacBio HiFi reads offer the dream-team combination of high accuracy and long read lengths. A typical HiFi read is 15-20 kb in length with 99.9% accuracy. With the length needed for reference-quality assemblies, plus accuracy that is comparable to or even better than short reads, HiFi reads are valuable for assembly of any genome of any size.

This combination of accuracy and read length also means you can create high-quality, haplotype-resolved genomes, making HiFi reads the first known technology to break through the phasing barrier using a single data type. Researchers around the world are leveraging HiFi sequencing to create haplotype-phased genome assemblies of multiple plant and animal species, even those with large, complex, and/or polyploid genomes.

Learn more about using HiFi reads for plant and animal genomics.

Myth #2:

PacBio sequencing is a luxury that is too expensive for large genomes, like the organisms I study.


This statement is outdated.

The launch of the Revio long-read sequencing system makes whole-genome haplotype-resolved sequencing much easier and more affordable than you might expect. You can now use HiFi reads to generate reference-grade assemblies for any organism—even complex species like the European mistletoe.

Each SMRT Cell on the Revio system can generate 90 Gb of HiFi data for about $1,000 USD. That means 30X coverage of a 3-Gb genome per Revio SMRT Cell. If you’re studying smaller species, flexible multiplexing options make it possible to sequence multiple samples together and further reduce the cost per genome.

Read more about how the Revio system makes genomic complexity a whole lot easier.

Myth #3:

Despite the better data quality of PacBio, its throughput and workflows can’t be scaled to the level I need to make the switch from short reads to HiFi sequencing.


This statement is outdated.

The Revio system can sequence 4 SMRT Cells in parallel, which means you can scale up quickly to run multiple applications at once. This capability can grow with you as your research evolves. Switch between tackling large and complex species or generating pangenomes for smaller species in a single day.

Image and text showing what you can do with one SMRT Cell

*Image from brochure

The high throughput of the Revio system means that high-volume applications, such as low-pass genotyping, can now take advantage of the benefits of HiFi reads. Researchers can identify complex structural variants, or variants in challenging regions of the genome that would otherwise be missed with short-read technologies, and then use that information to make informed decisions and enhance breeding programs.

Myth #4:

Even if I wanted to switch to HiFi sequencing, the DNA requirements and library prep process don’t work for my sample types.


This statement is misleading.

You can extract DNA and sequence any species with PacBio—just use the Nanobind PanDNA Kit, compatible with virtually any plant or animal species.

PacBio offers end-to-end workflows, from DNA extraction through sequencing, that are compatible with our automation partners. These workflows can flex from single-tube to 96-plate formats to match your throughput needs. It doesn’t end there—the new HiFi prep kit 96 reduces costs by 40% and cuts workflow time by 60% compared to prior kits. Now it’s possible to create 96 libraries for long-read sequencing in just 13 hours.

But what about data analysis? Workflow management in SMRT Link provides fast, easy, and powerful analysis of HiFi sequencing data from the Revio system. PacBio has built comprehensive whole-genome sequencing analysis tools that are ready to go in SMRT Link. Use Genome Assembly, available directly within SMRT Link, to generate high-quality (>Q50) phased de novo assemblies.

In addition, HiFi sequencing generates dramatically smaller data files compared to other long-read technologies. The average file size coming off the Revio system, for example, is only 55 Gb, a fraction of the ~1 Tb generated by alternate long reads. This smaller file size means analysis is less intensive, less computationally expensive, and ultimately faster.

“The size of [PacBio] data was a huge advantage, because it saved computational time and resources.”
— Dr. Carolina Jaramillo Oquendo, University of Southampton

Myth #5:

Getting epigenome information means doing extra library prep and costly additional sequencing, which I can’t afford.


This statement is false.

PacBio systems offer the unique ability to sequence native DNA, with all epigenetic markers and modifiers intact. That means that you can sequence the genome and epigenome together and detect 5mC methylation at CpG sites with a standard HiFi WGS workflow. No extra sequencing. No additional costs.

By detecting 5mC as a fifth base in addition to A, C, G, and T, HiFi sequencing allows methylation calling in complex and repetitive regions of the genome that are missed by other methylation assays—including microarrays, short-read sequencing, and nanopore sequencing.

Myth #6:

PacBio RNA sequencing is only useful for discovery applications, like annotation and isoform discovery, due to high costs and low throughput.


This statement is incomplete.

With high-throughput RNA sequencing on the Revio system, you can now do bulk and single-cell RNA sequencing for counting applications with PacBio.

The future of RNA sequencing is long reads. The Iso-Seq method—PacBio long-read sequencing solution for RNA studies— has already led to a wide range of discoveries in plant and animal biology, enabling investigators to study agriculturally important crops, endangered species, model organisms, and more.

New high-throughput RNA sequencing kits from PacBio supercharge this ability. Combining the Iso-Seq method with new Kinnex kits—the MAS-Seq method—further increases throughput, for both bulk and single-cell RNA sequencing.

Table outlining the advantages of full-length RNA sequencing through PacBio

*Image from whitepaper

For more information, read the whitepaper: Transforming plant and animal research with full-length and single-cell isoform sequencing.

Tomorrow’s genomic discoveries start today

The power of HiFi long reads means that researchers who study large or complex genomes need no longer be limited by short-read technology. Now, you can take advantage of read lengths and accuracy that allow you to see the full picture of the species you study. Learn more about how PacBio sequencing can help you tackle today’s challenges in plant and animal genomics for a brighter, more sustainable future.

Stay tuned for part five in our six-part series, where we disprove common myths about long reads in microbiology. Let the myth-busting continue!

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