April 4, 2024  |  HiFi difference

Long-read sequencing myths: debunked.
Part 1- HiFi sequencing

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In the fast-evolving landscape of genomics, the pace can be dizzying, and the fear of falling behind is real. Genomic research undergoes constant change, so if you want to keep your finger on the pulse of what’s available, you need to know the facts. Not too long ago, long-read sequencing was an emerging technology. Today, PacBio HiFi long reads are routinely used across the world to sequence thousands of genomes, epigenomes, and transcriptomes. HiFi sequencing applies to projects large and small, from academic research endeavors to large-scale population health studies.

The rate of technological change has been so dramatic that perceptions about the accuracy, uses, cost, and convenience of long-read sequencing are often out of step with the reality of what is true today.

Follow us in this six-part series, which will dispel common myths about long-read sequencing. Part one of the series focuses on common misconceptions around PacBio HiFi sequencing.

Prepare to be provoked, to be challenged, and to forget the dusty old textbooks with “that’s-how-it’s-always-been-done” attitudes.

Myth #1:

PacBio long-read error rates are too high.


This statement is outdated.

Many scientists first learned about long-read sequencing with the launch of the PacBio RS in 2011. That system stood out with its ability to generate reads of 1-10 kb, but had a major downside: a raw error rate of around 10%. Accuracy remains a challenge for long-read nanopore technologies, but PacBio sequencing has made extraordinary leaps in chemistry and sequencing methodology. In 2019, PacBio introduced HiFi sequencing, which achieves a typical accuracy of 99.9% (0.1% error rate) for 15–20 kb reads.

Today, this accuracy is a differentiating strength. HiFi reads offer the same read accuracy associated with short-read sequencing with the added benefit of length needed to span genomic repeats. HiFi reads have been used to generate a human genome with less than one error per million bases and to identify mutations in individual DNA molecules. A series of technology benchmarks, including the precisionFDA Truth Challenge v2 and the ABRF Next-Gen Sequencing study have shown HiFi sequencing to be the most accurate technology across many dimensions.

Myth #2:

HiFi sequencing is too expensive compared to other sequencing technologies.


This statement is oversimplified.

Original long-read sequencers gained a reputation as expensive to obtain and to run; it cost many thousands of dollars to sequence a human genome in the 2010s and even small microbial genomes were considered expensive due to library prep costs. But continuous innovation has changed that.

The Revio system, which launched in 2022, brought the sequencing cost of a 30× HiFi long-read human genome to $995 USD.* Improvements in the library prep workflow, like automated pipette shearing, and the introduction of new high throughput HiFi prep kits have brought the costs of preparing samples down to ~$56 for a human genome, and ~$39 for a microbial genome. Multiplexing samples can further reduce sequencing costs:

• Full-length transcriptomes at 5M reads for ~$125 per transcriptome (8 samples per SMRT Cell)
• Microbial genomes for ~$3 per genome (384 samples per SMRT Cell)

HiFi sequencing may be more expensive per Gb than large scale short-read sequencing platforms, but it matches costs of popular mid-throughput platforms. And, more importantly, the information provided by HiFi sequencing is richer, delivering a lower cost-per-answer compared to other sequencing technologies. While some short-read sequencing methods may offer an affordable base-for-base cost, they are limited in scope and usually require additional follow-on tests to achieve structural variant detection, phase genomes, and resolve coverage uniformity issues. Each of these additional methods come with their own time and labor costs, and those costs add up.

In contrast, HiFi sequencing allows researchers to capture the whole picture of genetic variation with a single assay and arrive at results for a considerably lower total cost compared to other sequencing technologies.

Myth #3:

HiFi sequencing is low-throughput and doesn’t scale effectively.


This statement is false.

Scale is another area where long-read sequencing has changed dramatically. Each generation of PacBio systems has supplied an order-of-magnitude increase in scale. The Revio system brought a 15x throughput leap compared to the Sequel IIe system. It delivers more complete, phased human genomes at a scale that even large commercial laboratories and service providers can use; output of 360 Gb per day for up to 1,300 human genomes per year. Using the Revio system, labs can scale up projects, sequencing up to ~3,900 de novo plant/animal genome (1 Gb) assemblies, ~5,200 Kinnex full-length RNA samples, or ~1,300 Kinnex single-cell transcriptomics samples per year.

Myth #4:

HiFi sequencing sample prep and analysis are laborious, time consuming, and don’t integrate well with existing laboratory infrastructures.


This statement is outdated.

Sample shearing, HiFi library prep, and analysis solutions are now quicker and easier than ever. It only takes ~6 hours (plus QC) to prepare 8 samples using the SMRTbell prep kit 3.0 or ~13 hours to shear and prepare 96 samples for HiFi genome sequencing, all with automation options.

After sequencing, PacBio analysis solutions are available for every step of the workflow, from variant calling to phased de novo genome assembly. Tools like the PacBio WGS Variant Pipeline are available for secondary and tertiary analysis, from alignment to annotation. In addition, PacBio Compatible partners, as well as additional community-supported analysis tools, make it easy for those new to HiFi sequencing to get started.

Myth #5:

Long-read sequencing is a niche technology that’s only worthwhile for de novo genome assemblies and detecting complex structural variants.


This statement is false.

Lauded as Method of the Year in 2022, long-read sequencing is no longer a niche technology. Instead, it is quickly becoming the method of choice for researchers to access a more complete view of biology and detect virtually all types of variants with one test. PacBio HiFi sequencing on the Revio system brings versatility and multiomic exploration to every run. The system allows you to simultaneously run four SMRT Cells in a single sequencing run, meaning scientists can utilize one cell for WGS, one cell for RNA, one cell for single-cell RNA, and one cell for amplicons — all at once, with methylation calling in every sequencing run. This not only opens doors to more ‘omes’ at once, but also gives labs the ability to diversify the types of samples being run in tandem (human, plant, animal, microbials and more).

As demonstrated by its 1,000+ peer-reviewed publications in 2023 alone, the powerful capabilities of HiFi sequencing continue to be recognized. With 1,200+ PacBio sequencers placed in more than 40 countries, long-read sequencing is clearly not a niche technology, but rather the method of the future.

Tomorrow’s genomic discoveries start today

There it is — your starter pack for debunking HiFi sequencing myths! As bright minds leading the frontier of discovery, keeping ahead of how sequencing technology works means that you’re ahead of the curve. And with HiFi long reads and platforms like the Revio system, the possibilities for groundbreaking new discoveries are virtually limitless.

Stay tuned for part two in our six-part series, where we disprove common myths about long-read sequencing in human genomics applications. Let the myth-busting continue!

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*US list price is $995 for sequencing reagents for one Revio SMRT Cell, which has an expected yield of 90 Gb, equivalent to a 30× human genome

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