Application brief

TAXONOMIC AND FUNCTIONAL PROFILING
WITH HIFI METAGENOMICS

High-accuracy long-read sequencing to characterize complex
microbial communities
HiFi metagenome profiling provides more (and richer) functional information and precise
species classifications.
• Each HiFi read typically has an average of eight intact genes per read, providing richer information about genes thatco-occur in a single microbe, and may be co-expressed• 80-90% of HiFi reads have at least one functional annotation, and typically average four, whereas only ~1/3 of shortreads can be annotated even once
• HiFi reads can assign taxonomy with high precision and recall

HiFi datasets
Average precision vs average recall
Species level(B)
1.0

1.0

0.4

0.2

0.0
0.2

0.4

0.6Precision

0.8

1.0

0.4

0.2

654
3202

100

10Sample 1a Sample 1b Sample 2a Sample 2b Sample 3a Sample 3bPercent of annotated HiFi reads

0.0

C

0.0

0.6

6050
4030

Average annotations per HiFi readaken
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0.8

80
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Recall0.8

F1

MethodKraken2
BrackenCentrifuge-h22
Centrifuge-h500
Metaphlan3
mOTUs
Sourmash-k31
Sourmash-k51
Metamaps
MMseqs2
MEGAN-LR-Prot
MEGAN-LR-Nuc-HiFi
MEGAN-LR-Nuc-ONT
BugSeq-V2

HiFi Datasets
100
Average F1 Score
Species90
Level

Percent annotated(A)Shotgun metagenome functional profilingNumber of annotations

Shotgun metagenome taxonomic profilingWith highly accurate long-read metagenomic data, you can profile species
with high precision and recall; tools/methods have been evaluated toprovide recommendations to which work best for long-read metagenomic
data.1

With an average of eight complete genes per HiFi read, PacBio® data
provides rich functional information; nearly every read contributes tothe understanding of the biological functions present in the microbialcommunity.²

Typical workflow from DNA to metagenome taxonomic and functional resolutionPrepare
SMRTbell® template

SMRT® sequencing andSMRT® analysis

Tertiary
data analysis

Create ~10–15 kb libraries for
HiFi metagenome sequencing3

Obtain HiFi reads with theSequel® II/e or Revio™ system

Profile metagenome taxonomy andfunctions using our GitHub pipelines5
with Sourmash6 or Diamond7 +MEGAN-LR8, or with the BugSeq
cloud-based platform9“Our comparisons between long-read sequencing technologies indicate that read quality
remains critical for taxonomic profiling performance…The long-read datasets produced
significantly better results than the short-read datasets.”1– Portik, Brown, and Pierce-Ward, 2022

The PacBio partnership with BugSeq
empowers public health labs to solve
complex microbial questions
Yield top accuracy for taxonomic profiling

Enable strain-level abundance

For the best precision/recall trade-off for taxonomic profiling, use BugSeq
with HiFi reads.1 BugSeq offers a curated reference database for analyses
requiring the highest quality, and the NCBI nucleotide database for broader
taxonomic representation.

In a comparison of analytical tools, BugSeq produced the most accurate
abundance estimates and classified the greatest number of reads to thestrain rank using PacBio HiFi data.1

Dive deep and analyze antimicrobial resistance

Discover with simplicity
With HiFi data, it’s as easy as uploading raw data. BugSeq is the only tool
tested by Portik et al. (2022) that is cloud-based, has a graphical userinterface, and performs quality control.1“Overall, we find that BugSeq, MEGAN-LR-prot, andMEGAN-LR-nuc provide the best tradeoffs for all
long-read metagenomics data.”1– Portik, Brown, and Pierce-Ward, 2022
Demo antimicrobial resistance report from BugSeq outputs.
Beyond taxonomic profiling, BugSeq performs de novo assembly andidentifies predictors of antimicrobial resistance (genes and mutations) as
well as virulence factors using curated databases.10

Learn more about PacBio microbial community applications and BugSeq:
pacb.com/metagenomics | bugseq.com/pacbio

KEY REFERENCES
1. Portik, D.M., et al. (2022) Evaluation of taxonomic classification and profiling methods for long-read shotgun metagenomic sequencing datasets. BMC Bioinformatics,
23: 541. 10.1186/s12859-022-05103-0
2. Gehrig, J., et al. (2022) Finding the right fit: evaluation of short-read and long-read sequencing approaches to maximize the utility of clinical microbiome data.
Microbial Genomics, 8(3), 10.1099/mgen.0.000794.
3. Procedure & checklist – Preparing whole genome and metagenome libraries using SMRTbell prep kit 3.0. PacBio documentation.
4. Application brochure – A new paradigm: HiFi reads for highly accurate long-read sequencing. PacBio documentation.
5. https://github.com/PacificBiosciences/pb-metagenomics-tools
6. Pierce, N. T., et al. (2019) Large-scale sequence comparisons with sourmash. F1000Research, 8, 1006. 10.12688/f1000research.19675.1
7. Buchfink, B., et al. (2015) Fast and sensitive protein alignment using DIAMOND. Nature methods, 12(1), 59–60. 10.1038/nmeth.3176
8. Huson, D. H., et al. (2018) MEGAN-LR: new algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs.
Biology direct, 13(1), 6. 10.1186/s13062-018-0208-7
9. https://bugseq.com/ & https://bugseq.com/pacbio
10. Chandrakumar et al. (2022) BugSplit enables genome-resolved metagenomics through highly accurate taxonomic binning of metagenomic assemblies.
Communications Biology, 151(5). 10.1038/s42003-022-03114-4.
Research use only. Not for use in diagnostic procedures. © 2023 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved. Information in thisdocument is subject to change without notice. PacBio assumes no responsibility for any errors or omissions in this document. Certain notices, terms,conditions and/or use restrictions may pertain to your use of PacBio products and/or third-party products. Refer to the applicable PacBio terms and conditionsof sale and to the applicable license terms at pacb.com/license. Pacific Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq,Sequel, Nanobind, SBB, Revio, and Onso are trademarks of PacBio.

102-326-574 REV01 16MAY2023