May 16, 2024  |  Microbiome

Long-read sequencing myths: debunked.
Part 5 — microbiology

Header image with microbes in the background and HiFi Facts logo

The organisms you study might be tiny, but the importance of the work you do is anything but. PacBio HiFi sequencing brings the microscopic world into sharp focus, offering the precision and long read lengths essential for pivotal work in areas like public health, pathogen surveillance, infectious disease research, drug development, and environmental cleanup.

Curious about what HiFi accuracy can actually do for your microbiology and metagenomics research? Chances are, it’s much more than you think. Imagine pinpointing community composition down to the species or strain level, capturing complete 16S rRNA sequences, or producing hundreds of top-notch metagenome-assembled genomes (MAGs)—just for starters.

This is part five of our six-part myth-busting series, where we dispel common myths and misconceptions about PacBio sequencing in microbial genomics applications.

Myth #1:

16S sequencing doesn’t give me species-level resolution, and long-read sequencing has too many errors. For genome assembly, my best option is to use a hybrid approach of short and long reads.


This statement is inaccurate.

Now, you can get species-level resolution with 16S when using just one sequencing method: full-length 16S sequencing with HiFi long reads, which analyzes the full length of the target gene. This unbiased approach boasts a high rate of reads that can be classified down to the species level—and even strain level—with accuracy.

A 2023 study compared three different approaches for microbiome taxonomic classification, including short reads and full-length sequencing with PacBio long reads. Sure enough, full-length 16S sequencing showed the highest discriminating power, beating short reads.1

Which leads us to ask an important question: why settle for guessing with partial 16S short-read sequencing when full-length 16S long-read sequencing offers the clearest picture? In addition, HiFi reads give you the read length and accuracy you need for assembling high-quality genomes from the get-go, making short-read polishing a thing of the past.

“With [PacBio], we can now sequence complete genomes of nearly all abundant bacteria in a microbiome. Short-read studies rarely provided complete sequences of even a single microbe.” 2
— Mikhail Kolmogorov, National Cancer Institute

Myth #2:

PacBio HiFi sequencing is too expensive for microbial sequencing compared to short reads.


This statement is outdated.

The combined power of the latest Kinnex and HiFi plex prep kits enables new capabilities in microbiology that are downright futuristic. The Kinnex 16S rRNA kit is a versatile throughput boosting solution based on the MAS-Seq method that takes advantage of very long HiFi read lengths to concatenate multiple full-length 16S amplicons into single molecules which yields up to 12-fold more 16S reads per sample. Engineered for efficiency, the new HiFi prep kit 96 is designed to allow users the ability to automate long-read sequencing workflow steps and streamline the process of preparing, pooling, and loading samples, at a significant reduction in cost.

Using the Kinnex 16S rRNA kit, HiFi plex prep kit 96, and the Revio system together gets you a cost per sample of ~$5 for full-length 16S sequencing. For shotgun metagenomic profiling and microbial isolate whole-genome sequencing, the cost per sample is ~$50, including library prep and sequencing reagents (excluding DNA extraction).

PacBio HiFi sequencing is now as affordable as short reads
across all microbial genomics applications


Cost per sample  ILMN NextSeq 2000* PACB Revio** PACB HiFi advantage 

384 smp: $11

768 smp: $6

1,536 smp: $4  

384 smp: $6

768 smp: $5

1,536 smp: $4 

Accurate full-length 16S yields one of the best possible species and strain resolution for microbiome samples 
Shotgun metagenome profiling 

48 smp: $103

96 smp: $92 

48 smp: $60

96 smp: $49 

Accurate long reads yield high precision and recall for taxonomic classification as well as richer functional annotations with 80-90% of HiFi reads being classified and annotated 
Microbial WGS 

96 smp: $67

384 smp: $39 

96 smp: $49

384 smp: $42 

Accurate long reads yield closed chromosomes and accessory plasmids + methylation signatures 

*Cost per sample pricing for ILMN NextSeq 2000 is calculated using ~$30 per sample library prep and NextSeq 2000 P2 and P3 300 cycle sequencing reagent and flow cell list prices found on website.
**Cost per sample for PACB 16S is calculated using the Kinnex 16S rRNA kit and Revio sequencing reagents. Cost per sample for PACB shotgun metagenome profiling and microbial WGS are calculated using the HiFi plex prep kit 96, SMRTbell adapter index plate 96A, and Revio sequencing reagents.


Myth #3:

It’s too hard to scale up PacBio sequencing for microbial whole-genome sequencing, 16S, and shotgun metagenomics.


This statement is outdated.

The new Revio system from PacBio changes the game, delivering a 15X increase in HiFi read throughput compared to previous Sequel IIe systems. That translates to ~15,600 shotgun metagenome assemblies per year, and 360 Gb of HiFi reads per day.

With the new HiFi plex prep kit 96 and Kinnex 16S rRNA kit, the capabilities of Revio continue to grow, so you can keep doing more with less investment up front and scale up microbial WGS, full-length 16S, and shotgun metagenomics profiling projects.

Table showing samples and SMRT Cells for microbial applications

See how it’s done for 16S by reading the application note: Kinnex 16S rRNA kit for full-length 16S sequencing.

Myth #4:

PacBio prep workflows are cumbersome and long-read analysis tools are limited.


This statement is incomplete.

Prep is now easier—and faster—than ever. The new HiFi plex prep kit dramatically accelerates microbial long-read workflows, so you can prep 96-384 samples in 6 hours for a single Revio SMRT Cell, or up to 1,536 samples in a single Revio run. The HiFi plex prep kit does all this while slashing costs by more than 50%, so you can get more long-read data for your sequencing dollars.

PacBio also offers end-to-end, automatable workflows for popular microbial applications, including WGS, shotgun metagenomics, and full-length 16S sequencing. These protocols accelerate the whole process for PacBio long reads, especially shrinking the up-front extraction and library prep steps. Check out how the rapid HiFi microbial WGS workflow works and see whether your automation options are already verified by PacBio.

Curious about automation and analysis? Try the 16S and metagenomics analysis workflows in GitHub or download some sample Kinnex 16S or metagenomics data and see for yourself.

Myth #5:

Metagenome-assembled genomes (MAGs) aren’t practical to do with HiFi sequencing.


This statement is false.

PacBio HiFi sequencing recovers more high-quality MAGs and more circular, single-contig MAGs than other technologies, even at lower coverage. With an average read length of up to 20 kb and accuracy above Q30, HiFi sequencing gives you the contiguity and completeness necessary to generate reference-quality MAGs. HiFi sequencing also achieves a cost per MAG of under approximately $5 USD—an order of magnitude cheaper than the cost of microbial single-isolate WGS. What other technologies can do both?

“HiFi reads allows us to generate a nearly complete picture of the metagenome, not just a fragmented assembly. Like complete genomics, which is already being applied to rare disease diagnostics, complete metagenomics may soon make its way into medicine and many other disciplines.” 2
— Pavel A. Pevzner, University of California San Diego

PacBio recently collaborated with Zymo Research, Phase Genomics, and others to generate MAG datasets from the ZymoBIOMICS Fecal Reference with TruMatrix Technology (D6323). You can download the PacBio datasets and check out the preprint to see the HiFi difference for yourself.

Learn more about the performance enhancements you can get by using HiFi metagenomics on the Revio system.

Tomorrow’s genomic discoveries start today

As we’ve shown above, PacBio HiFi sequencing really is setting new standards in metagenomics and microbiome sequencing, giving scientists the flexibility and high resolution they need to characterize complex microbial communities, identify novel species, and more. We encourage you to reimagine what’s possible in microbial genomics with HiFi reads on the Revio system.

Stay tuned for part six in our series, where we wrap up by disproving common myths about long reads in cell and gene therapy research. Let the myth-busting continue!

New to the series? Check out the other installments:
Part 1 – HiFi sequencing
Part 2 – human genomics
Part 3 – cancer genomics
Part 4 – plant and animal genomics

Are you ready to try HiFi?

Take advantage of our spring promo


  1. Notario E, Visci G, Fosso B, et al. Amplicon-Based Microbiome Profiling: From Second- to Third-Generation Sequencing for Higher Taxonomic Resolution. Genes (Basel). 2023 Jul 31;14(8):1567. doi: 10.3390/genes14081567. PMID: 37628619; PMCID: PMC10454624.
  2. Baxt, J. (2022). Long-reads and powerful algorithms identify “invisible” microbes

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