Metagenomics
Generating new insights into how microbial communities function and interact with their environments requires improved approaches. Long-read sequencing enables species-level resolution and drives functional insights with full-length 16S sequencing and 10 kb HiFi reads for metagenome profiling and assembly.
Explore the microbial world with confidence
Highly accurate long reads – HiFi reads – with single-molecule resolution are ideal for full-length 16S rRNA sequencing, shotgun metagenomic profiling, and metagenome assembly, so that you can:
- Determine community composition at the species or strain-level with competitively priced full-length16S sequencing
- Identify 6-8 full-length genes in every HiFi read with efficient, cost-effective metagenomic profiling
- Generate up to 20 high-quality metagenome-assembled genomes (MAGs) per multiplexed human fecal sample with just 6 Gb of HiFi data
- Leverage epigenomic data to associate contigs and plasmids from closely related strains
Workflow: from DNA to resolved microbial communities
Library preparation
Prepare targeted sequences or shotgun libraries to characterize your microbial community.
Explore recommendations for all SMRT sequencing applications
Extract DNA using the commercially available kit of your choice
Full-length 16S sequencing
- Use our full-length rRNA protocol available with recommended primers
- Or use Shoreline Biome Complete StrainID or V1-V9 kits for DNA extraction and PCR amplification
Shotgun metagenomic profiling and metagenome assembly
- Create 10 kb libraries for HiFi metagenomics
Sequencing
Use the Sequel II or IIe systems for affordable, high-quality metagenomic sequencing
Full-length 16S sequencing
- With 1.6 kb HiFi reads generate up to 3.5 million >Q20 reads with a 10-hour movie collection time
- Resolve up to 192 communities per SMRT Cell 8M
Metagenome profiling
- With 10 kb HiFi reads generate up to 2.4 million >Q20 reads with a 30-hour movie collection time
- Profile up to 8 communities per SMRT Cell 8M for taxonomic or functional profiling
Metagenome assembly
- With 10 kb HiFi reads generate up to 2.4 million >Q20 reads with a 30-hour movie collection time
- Multiplex up to 4 samples per SMRT Cell 8M to generate high-quality MAGs
*Read lengths, reads/data per SMRT Cell 8M and other sequencing performance results vary based on sample quality/type and insert size.
Data analysis
Make discoveries using bioinformatics tools in SMRT Analysis or PacBio DevNet.
Full-length 16S sequencing
- Use circular consensus sequencing analysis to produce HiFi reads through multiple observations of single circularized templates
- Analyze 16S date with either Shoreline Biome SB Analyzer or DADA2
Metagenome profiling
- Use HiFi sequencing to study metagenome compositions and functions using DIAMOND and MEGAN-LR with our GitHub-posted pipeline.
Metagenome assembly
- De novo assemble metagenomes to high consensus accuracies >99.999% with hifiasm-meta or HiCanu
- Bin high-quality MAGs from HiFi assemblies with our streamlined, GitHub-posted pipeline using minimap2, MetaBAT2, CheckM, and GTDB-Tk
- Utilize epigenetic signatures to improve shotgun metagenomic analysis
Application brief
Metagenome sequencing with HiFi reads
Learn more these best practices for metagenomic sequencing with HiFi reads
Spotlight
Strain-level identification of bacterial communities with the unprecedented accuracy of PacBio full-length 16S sequencing
Using DADA2 software, all copies of the 16S housekeeping gene can be recovered from microbial communities. 16S sequence variants from the same strain appear in the data in integer ratios that reflect their copy number in each genome. In some cases, for example E. coli O157:H7 in the Zymo mock community, this high-resolution information can be used to unambiguously identify not only the species but the strain present. Explore this research further:
Callahan, B. J., et al. (2019). High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution. Nucleic Acids Research.
Spotlight
Both read length and accuracy matter for metagenome assembly and functional profiling
Comparing analysis results using HiFi data from a sheep fecal sample to results using lower accuracy subreads from the same data set show the impact of improving accuracy on assembly contiguity, MAG recovery and gene discovery. In the figure above, the more contiguous HiFi assembly enabled recovery of 25% more complete biosynthetic gene clusters (BCGs) than noisy CLR long reads. Explore this research further:
Bickhart, et. al. (2021). Generation of lineage-resolved complete metagenome-assembled genomes by precision phasing. BioRxiv. https://doi.org/10.1101/2021.05.04.442591.
