Shotgun Metagenomics with HiFi Playbacks: Now More Affordable

Unlocking the secrets of complex communities and microbiomes represents one of the most exciting frontiers in microbiology, with the potential to revolutionize our understanding of human health, food production and environmental preservation. To lay the groundwork for such breakthroughs, it is essential to fully capture and profile all the genetic information present in a complex microbiome or community; and shotgun metagenomic methods are essential for obtaining such information.

In search of the best shotgun metagenomic data, PacBio HiFi sequencing continues to set the standard for quality and completeness, thanks to exciting innovations in data analysis tools. These new pipelines, together with the powerful combination of high accuracy and long read-time of HiFi data, provide more accurate profiling information than any other technology on the market. Plus, HiFi data returns more assembled single-contig circular metagenome (MAG) genomes than ever before, giving you the power to discover the hidden functions of the microbial ecosystem and confidently capture the richness and diversity of species.

In one look

  • HiFi shotgun metagenomic sequencing generates datasets consisting of individual reads spanning up to 8 contiguous genes, allowing you to precisely profile the function and taxonomy of a community, all at once.
  • Taxonomic and functional profiling of HiFi metagenomes can now be performed at nearly one-third the cost† without compromising accuracyusing information from subsampling experiments.
  • Assembly-focused HiFi metagenomic studies can return tens to hundreds of high-quality (HQ) MAGs, with up to 33% of those MAGs being a single contig, allowing you to predict genes and make taxonomic annotations with authority.

Get stunning quality HiFi metagenomic profiling data at lower cost


Profile more affordably using just 0.5GB of HiFi data to get the same state-of-the-art species and diversity information that can be found with 88GB, or probably more.

The proof is in the research: In order to help microbiologists obtain the most cost-effective information possible from HiFi data, we performed a series of subsampling studies. Each analysis was performed to find the minimum depth needed to obtain highly accurate community profiles as well as to determine recovery rates for MAG and single-contig MAG.

To begin, we sequenced the ZymoBIOMICS fecal reference with TruMatrix technology, which is a pooled highly complex human gut microbiome sample that is extremely useful for benchmarking and reference standards. We sequenced this sample using 4 SMRT 8M cells, then took the dataset and downsampled it incrementally to coverage equivalent to a multiplexed sequencing sequence of 96 on a single SMRT cell. 8M (96-plex). The study captured a total range of 88 to 0.3 gigabases (Gb) of data. Each level of data was then run through PacBio’s taxonomic profiling workflow.

HiFi data captures abundance at shallow depths
Figure 1.) The taxonomic results of subsampling with the optimized filter in the PacBio metagenomic profiling pipeline (described in this webinar). The number at the top of each bar is the total number of species detected at each level of data and the colors represent relative abundances.

The results of this workflow showed that the information obtained from 4 SMRT 8M cells up to 48-plex is largely consistent, with not only similar numbers of species recovered (Fig. 1), but the profiles of relative abundance also being nearly identical (Figure 2). This indicates that the same level of taxonomic profiling information can be obtained with 0.5 GB at the 48-plex level as 88 GB, providing microbiome researchers with an even more affordable entry point into the world of HiFi metagenomics.

Species richness information
Figure 2) Relative abundance and taxonomic assignments for the ZymoBIOMICS TruMatrix fecal reference, subsampled to progressively lesser sequencing depth starting at 4 SMRT cells and ending at 96-plex.

Listen to Dan Portik Ph.D. (@DPortik) Senior Scientist, Bioinformatics at PacBio, discusses these findings in our recent metagenomics webinar, on demand now.

Capture species in exquisite detail with more HQ-MAG than ever before


Achieve optimal cost-benefit ratios from one sample per 8M SMRT cell for assembly-focused metagenomic HiFi sequencing. Projects seeking deeper insight can increase sequencing depth with multiple 8M SMRT cells to capture rare microbes in stunning detail.

How do we know: The second half of our subsampling study was devoted to establishing guidelines for metagenome assembled genome (MAG) studies. To assess the effects of sequencing depth on MAG assembly, we took the ZymoBIOMICS TruMatrix 4 SMRT Cell 8M gut microbiome dataset and progressively downsampled it to a depth of 8 plexes to examine the effects on assembly and recovery at each level. We know that assembly is much more sensitive to sequencing depth and that there is a very predictable relationship between sequencing depth and outcome. However, in our pipeline, depth has a different impact on HQ-MAG recovery and single-contig MAG recovery.

HQ-MAG recovery
Figure 3.) A comparison of the number of assembled genomes per metagenome (MAG) retrieved versus depth of sequencing. The green line shows the total number of high-quality (HQ) MAGs returned and the purple line shows the number of single-contig MAGs retrieved at a given data level.

The results of our workflow show that from 8-plexes there is a logarithmic relationship between total MAG recovery and depth (green line in Fig. 3), with rapid gains down to a single cell SMRT 8M. From here, recovery efficiency is somewhat reduced as sequencing depth increases, but HQ-MAGs are still returned in large numbers, with the deeper sequencing yielding what are almost certainly deeper species rare. For recovery of single-contig MAGs (Fig. 3, purple line), there is a distinct linear relationship between recovery and depth with 4 SMRT Cell 8M yielding 199 total HQ-MAGs, of which 72 are single-contig. Even at 8-plex depth, 9 HQ-MAGs were recovered and 2 were single contigs. At 4-plex, 34 HQ-MAGs were generated with 9 being single-contig. These results clearly indicate that HiFi shotgun metagenomic sequencing can provide many extremely detailed genome assemblies that would be difficult to obtain with alternative sequencing technologies.

Listen to Dan Portik Ph.D. (@DPortik) Senior Scientist, Bioinformatics at PacBio, discusses these findings in our recent metagenomics webinar, on demand now.

Observe the microbial world with stunning clarity


HiFi metagenomics gives microbiome researchers the power to accurately profile function and taxonomy of a community with greater profitability than ever before. At the same time, the incredible length and accuracy of HiFi readings allow investigators to take advantage of the impressive HQ-MAG recovery to predict genes and make taxonomic annotations with greater confidence. Together, these advantages make HiFi sequencing the premier sequencing solution for ambitious projects aiming for breakthrough discoveries.

Want more?

Check out the recap of our recent metagenomics webinar
Watch our HiFi metagenomics talk, available on demand now
Discover our HiFi solutions for metagenomics
Access HiFi Metagenomics Datasets
Try the PacBio HiFi metagenomics pipeline


Compared to the previous recommendation of 8 samples multiplexed by SMRT Cell 8M. The 48-plex shotgun metagenome profiling recommendation is for community standard taxonomic and functional profiling using optimized PacBio github pipelines. If you are interested in more in-depth functional profiling analysis, you can target less multiplexed samples on a single 8M SMRT cell.

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