Sample Submission Guidelines
Why PacBio HiFi — Accuracy That Short Reads and Other Long Reads Can't Match
If you work with Illumina data, you know the trade-off: high per-base accuracy, but reads too short to span repeats or resolve isoforms. If you've explored nanopore sequencing, you've gained length — but at the cost of raw accuracy. PacBio HiFi is the only platform that eliminates this compromise.
What happens inside a ZMW
PacBio Single Molecule, Real-Time (SMRT) sequencing takes place inside Zero-Mode Waveguides — nanoscale observation chambers where a single DNA polymerase incorporates fluorescent nucleotides one at a time. Unlike nanopore sequencing (which infers bases from ionic current) or Illumina (which builds reads from clusters of identical fragments), SMRT watches a single molecule in real time.
The HiFi difference: SMRTbell adapters circularize each DNA molecule. The polymerase then reads around the circle multiple times. These multiple passes are combined into one HiFi consensus read — giving you both the length of a long read and the accuracy of multiple independent observations.
PacBio SMRTbell → ZMW → circular consensus → HiFi read with QV ≥30
Three capabilities you get from one SMRT Cell
- QV ≥30 long reads (~15–20 kb) — accurate enough for variant calling, long enough for assembly and phasing.
- Simultaneous 5mC methylation — polymerase kinetics reveal modified bases during the same run. No bisulfite. No separate library.
- Full-length isoforms without assembly — Iso-Seq/Kinnex captures each transcript from transcription start site to polyA tail as a single read. The isoform is measured, not reconstructed.
Where HiFi beats the alternatives
- vs Illumina short reads: Spans repeats, SVs, and full isoforms. Assembles complete genomes instead of fragmented contigs.
- vs Nanopore long reads: Single-molecule QV ≥30 accuracy with no polishing. Methylation detection built in, not retrofitted.
- vs running multiple experiments: One SMRT Cell gives you variants + methylation + isoforms. No parallel workflows to coordinate.
Our PacBio SMRT Sequencing Services
Every service below runs on the same HiFi platform — same accuracy, same methylation capability, same data quality.
Whole Genome de novo Sequencing
Reference-free genome assembly for microbial, plant, animal, and human genomes. HiFi reads span repeats and deliver contiguous assemblies with BUSCO completeness >90%.
Best for: Novel organism sequencing, pan-genome projects, T2T finishing.
HiFi-based human WGS for comprehensive variant detection — SNVs, indels, SVs, and phased haplotypes from one dataset.
Best for: Rare disease, SV discovery, pharmacogenomics, population studies.
Plant/Animal Whole Genome de novo
Multi-platform assemblies (HiFi + Hi-C + RNA-seq) for complex genomes including polyploids and large repetitive genomes.
Best for: Large genomes, polyploid species, agri-genomics, evolutionary biology.
Telomere-to-Telomere (T2T) Sequencing
Gap-free chromosome-scale assemblies using HiFi + ultra-long ONT reads. Captures centromeres, telomeres, and segmental duplications.
Best for: Reference-grade genomes, centromere biology, complete assembly finishing.
Full-Length Transcript Sequencing (Iso-Seq / Kinnex)
Complete isoform catalogs from TSS to polyA. Resolve splice variants, fusion genes, and APA — without short-read assembly ambiguity.
Best for: Isoform discovery, alternative splicing, fusion detection, transcript annotation.
Full-Length 16S/18S/ITS Amplicon Sequencing
Species-level taxonomic resolution with full-length rRNA gene amplicons. Accurate to the strain level where short amplicons stop at genus.
Best for: Microbial ecology, environmental samples, strain-level identification.
Long-Read Metagenomic Sequencing
HiFi metagenomics for MAG recovery, functional annotation, and strain-level community profiling. PacBio or Nanopore methods available.
Best for: Complex communities, MAG binning, functional gene discovery.
CiFi Sequencing | Pre-Made Library Sequencing | Long Amplicon Analysis (LAA)
Specialized workflows: CiFi for phased variant analysis, Pre-Made Library for customer-prepared SMRTbell libraries, LAA for targeted haplotype resolution.
Where PacBio HiFi Adds the Most Value
Below are the research areas where HiFi long reads solve problems that short reads or lower-accuracy long reads cannot. Each application maps to the right PacBio service.
De novo genome assembly & finishing
Span repeats and complex regions to produce complete, contiguous assemblies. Combine HiFi with Hi-C for chromosome-scale scaffolds.
Recommended services: De novo WGS, T2T Sequencing, Plant/Animal WGS.
Structural variant & rearrangement detection
Identify large insertions, deletions, inversions, and translocations with breakpoint-level resolution.
Recommended services: Human WGS, De novo WGS.
Haplotype phasing & allele-specific analysis
Long HiFi reads preserve linkage across distant variants — generate haplotype-resolved assemblies and variant calls.
Recommended services: Human WGS, CiFi Sequencing, LAA.
Full-length transcriptomics (isoforms & fusions)
Capture complete isoform catalogs from TSS to polyA tail — no computational assembly guesswork.
Recommended services: Iso-Seq/Kinnex Full-Length Transcript Sequencing.
DNA methylation (5mC) — simultaneous with sequencing
Genome-wide 5mC profiles from SMRT kinetics — no bisulfite conversion. One run = variants + methylation.
Recommended services: Any WGS service (methylation extracted from standard HiFi data).
Microbiome & metagenomics
Full-length 16S/18S/ITS for species-level taxonomy. HiFi metagenomics for MAG recovery and functional profiling.
Recommended services: Full-Length Amplicon, Long-Read Metagenomics.
Targeted sequencing & validation
Deep coverage of specific loci for variant phasing, repeat expansion analysis, and allele-specific characterization.
Recommended services: LAA, CiFi Sequencing, Pre-Made Library.
Pan-genome & population genomics
Build comprehensive pan-genome references with HiFi assemblies capturing SVs across populations.
Recommended services: De novo WGS, T2T Sequencing, Plant/Animal WGS.
What You'll Receive — Data Built for Publication
Every project ships with the raw data, processed results, and documentation you need to move straight to analysis — and straight into your manuscript.
| Data Category | What You Get | Why It Matters |
|---|---|---|
| HiFi Reads | FASTQ/BAM, QV ≥30, per-read QC tags | Ready for assembly, alignment, or variant calling — no polishing step needed |
| Subreads (BAM) | Raw polymerase reads with kinetic information | Enables methylation re-analysis and custom CCS parameter tuning |
| QC Report | Yield, read-length N50, Q-score distribution, CCS passes, barcode balance | Transparent evidence that your run met agreed-upon targets |
| Assembly | FASTA/GFA, N50/NG50, BUSCO completeness | Publication-ready genome with statistics reviewers expect |
| Variants | VCF with SNV/indel/SV, phased where applicable | Phased variants from long reads — no statistical imputation needed |
| Methylation | bedMethyl/TSV, DMR summaries, genome-wide tracks | 5mC calls from the same run as your variants — zero extra prep |
| Isoforms | GTF/GFF3 isoform catalog, expression tables, fusion list | Complete isoform models, each supported by full-length reads |
| Project Memo | Protocol, kit/chemistry details, software versions, run parameters | Everything a reviewer needs to evaluate your methods section |
How Your Project Runs — The PacBio SMRT Workflow
- Sample Intake & QC: Qubit + spectrophotometry. gDNA: A260/280 1.8–2.0, ≥30 kb modal length. RNA: RIN ≥8 for Iso-Seq.
- SMRTbell Library Prep: Adapter ligation, size selection per application. Kinnex concatenation for multiplexed Iso-Seq. Barcoded adapters for sample pooling.
- SMRT Cell Sequencing: Sequel II/IIe or Revio systems. Run times 10–30 hours depending on target yield and SMRT Cell type (8M or 25M).
- CCS → HiFi Generation: Subreads from each ZMW are processed through the CCS algorithm. HiFi reads require ≥3 full passes and meet QV ≥30 threshold.
- Analysis & QC: Assembly, variant calling, methylation detection, or isoform analysis per your selected service. Post-analysis QC with mapping rate and coverage metrics.
- Delivery: Structured data folders, full project memo, QC report, and optional results walkthrough with our bioinformatics team.
Sample Requirements
| Service | Amount & Integrity | Purity | Shipping | Notes |
|---|---|---|---|---|
| WGS (Microbial) | ≥1 μg gDNA, ≥30 kb | A260/280 1.8–2.0 | −20 °C | — |
| WGS (Plant/Animal) | ≥3–5 μg HMW gDNA, ≥30 kb | A260/280 1.8–2.0; A260/230 ≥2.0 | −20 °C | Wide-bore tips, no vortexing |
| WGS (Human) | ≥1–3 μg gDNA, ≥30 kb | A260/280 1.8–2.0 | −20 °C | — |
| T2T Genome | ≥3–5 μg HMW gDNA, ≥50 kb | A260/280 1.8–2.0 | −20 °C | Minimize all shearing |
| Iso-Seq / Kinnex | ≥500 ng–1 μg total RNA, RIN ≥8 | A260/280 ~2.0; A260/230 ≥2.0 | −80 °C (dry ice) | DNase if needed |
| 16S/18S/ITS Amplicon | ≥50–200 ng pooled amplicons | No primer dimers | 4 °C | Provide primer info |
| Metagenomics | ≥500 ng–1 μg gDNA | A260/280 1.8–2.0 | −20 °C | Note host depletion |
| CiFi / LAA | ≥1–2 μg HMW gDNA / ≥100 ng amplicons | Per application | −20 °C / 4 °C | Provide target regions |
| Pre-Made Library | ≥50 ng SMRTbell library | QC report required | −20 °C | We QC on arrival |
- General: Avoid phenol, heparin, EDTA, polysaccharides. Minimize freeze-thaw. Contact us for FFPE or low-input samples.
Bioinformatics — From HiFi Reads to Biological Insight
Every project includes standard bioinformatics. Advanced analyses are scoped to your specific research question.
- CCS & Demultiplexing: Subread → HiFi generation with QV filtering. Per-sample demux with barcode QC.
- De Novo Assembly: hifiasm or hicanu for contig-level assemblies. BUSCO completeness assessment. Optional Hi-C scaffolding for chromosome-scale assemblies.
- Variant Calling: DeepVariant / pbsv for SNV/indel/SV. Whatshap-based phasing using HiFi reads for haplotype-resolved VCFs.
- 5mC Methylation: pb-CpG-tools for per-site 5mC calling. DMR detection. Motif-specific methylation analysis. Genome-wide tracks.
- Isoform Analysis: Iso-Seq pipeline for isoform discovery, quantification, fusion detection, and alternative polyadenylation.
- Metagenomics: Taxonomic classification, MAG binning, functional annotation (KEGG, COG, CAZy).
Quality & Study Design
- Replicates: ≥2 biological replicates per condition. Spike-in standards available for quantification.
- Coverage: 30–60× HiFi for de novo assembly. Custom coverage modeling for variant detection and Iso-Seq based on your targets.
- QC Gates: HiFi yield, polymerase read N50, CCS pass count, and on-target fraction — all agreed before your run starts.
- DNA Quality: HMW gDNA ≥30 kb modal length. We verify integrity before library prep and alert you if input falls below threshold.
PacBio HiFi vs Nanopore vs Illumina
Choosing the right platform for your project? Here's how they compare on the dimensions that matter for research outcomes.
| What Matters | PacBio HiFi | Nanopore (ONT) | Illumina |
|---|---|---|---|
| Read accuracy (single molecule) | QV ≥30 (≥99.9%) | Improving; depth-dependent | ≥99.9% |
| Read length | ~15–20 kb HiFi; subreads longer | Up to Mb-class | 2×300 bp max |
| Methylation detection | 5mC from kinetics — same run, no bisulfite | 5mC/6mA from signal; Direct RNA | Not native |
| Isoform resolution | Full-length reads, no assembly needed | Full-length but lower base accuracy | Assembly-dependent (fragmented) |
| Best use case | Assemblies + variants + methylation from one dataset | Ultra-long span; time-sensitive or field work | Deep SNV/indel cohorts |
Quick decision guide
- Need highest long-read accuracy for assemblies and variants: choose PacBio HiFi.
- Need the longest molecules (telomeres, massive repeats): add Nanopore Ultra-Long.
- Need methylation + variants from one experiment: PacBio HiFi — no bisulfite, no extra prep.
- Large SNV/indel cohorts: Illumina for depth; add HiFi for SVs and phasing.
- Hybrid designs: HiFi + short-read or HiFi + ONT balances accuracy, completeness, and cost.
Actual performance depends on sample quality, library preparation, sequencing depth, and analysis pipeline.
Why CD Genomics for PacBio SMRT Sequencing
You have choices for PacBio service providers. Here's what we do differently.
- One run, more data: Our HiFi workflows are designed to maximize what you get from every SMRT Cell — variants, methylation, and assembly metrics from a single experiment.
- QC you can publish: Every project includes a detailed QC report with metrics reviewers expect. No black boxes.
- Analysis that matches your question: We scope bioinformatics to your research goals — not a one-size-fits-all pipeline.
- Cross-platform guidance: When HiFi alone isn't optimal, we'll tell you — and suggest a hybrid design that saves you money and time.
- SOP-driven, not ad-hoc: Every step — intake, library prep, sequencing, CCS, analysis — follows a documented SOP with defined acceptance criteria.
- Publication support: Need a methods paragraph, figure preparation, or reviewer response assistance? We've done this before.
- No instrument lock-in: We run PacBio Sequel II/IIe and Revio systems. You get the right SMRT Cell format for your project size — 8M or 25M.
Demo Results
HiFi Read Q-Score Distribution — median QV ≥30 across all passes
HiFi Read Length Distribution — N50 typically 15–20 kb per SMRT Cell
De Novo Assembly Metrics — contig N50, scaffold N50, and BUSCO completeness
PacBio SMRT Sequencing FAQ
1. What makes PacBio HiFi different from other long-read sequencing?
HiFi is the only long-read technology that delivers single-molecule QV ≥30 accuracy. It achieves this through circular consensus sequencing — each molecule is read multiple times and the passes are combined. By contrast, nanopore raw reads have lower per-base accuracy and typically require consensus or polishing steps. HiFi also detects 5mC methylation from polymerase kinetics in the same run, with no additional chemistry required.
2. How does HiFi accuracy compare to Illumina?
HiFi reads match Illumina's per-base accuracy (≥99.9%) while being 50–100× longer. This means you can call SNVs with the same confidence as short reads — but also detect structural variants, phase haplotypes, and assemble genomes without gaps. HiFi is not a trade-off between accuracy and length; it gives you both.
3. Can I detect methylation from my PacBio run?
Yes — and you don't need to do anything extra. PacBio polymerases slow down at methylated bases, producing kinetic signatures that the basecaller interprets as 5mC calls. These calls are generated during standard CCS analysis. No bisulfite conversion, no separate library, no parallel sequencing run. You get variants and methylation from one SMRT Cell.
4. What is Iso-Seq and when should I use it instead of short-read RNA-seq?
Iso-Seq (now also available as Kinnex for higher throughput) sequences full-length cDNA molecules from transcription start site to polyA tail in single reads. Use it when you need to identify complete isoform structures, detect fusion transcripts, or quantify isoform usage — tasks where short-read RNA-seq guesses at isoform assembly. Kinnex concatenates multiple transcripts per read, increasing throughput per SMRT Cell ~5-fold.
5. How much DNA do I need?
Standard WGS: ≥1–5 μg HMW gDNA at ≥30 kb modal length. T2T projects: ≥3–5 μg at ≥50 kb. Iso-Seq: ≥500 ng–1 μg total RNA at RIN ≥8. Lower inputs may be possible — contact us with your sample constraints.
6. How long does a project take?
Project timelines depend on library preparation, sequencing configuration, and bioinformatics scope. We provide a detailed timeline during project consultation based on your specific requirements.
7. What instruments do you use?
PacBio Sequel II/IIe and Revio systems, SMRT Cell 8M and 25M formats. All runs are monitored by experienced technicians with SOP-driven QC gates.
8. Can I send my own SMRTbell libraries?
Yes. Our Pre-Made Library Sequencing service accepts customer-prepared libraries. We QC on arrival, load onto the appropriate SMRT Cell, and deliver CCS data plus optional bioinformatics.
9. How is this different from the PacBio services I can get elsewhere?
Three differences: (1) We optimize each SMRT Cell for multiple data types — you get variants AND methylation AND assembly metrics from the same run. (2) Every project includes a publication-ready QC report with metrics reviewers expect. (3) We provide cross-platform guidance — if a hybrid HiFi + ONT or HiFi + Illumina design would serve your question better, we'll recommend it.
Case Study — Isoform-Resolution Analysis of Nonsense-Mediated mRNA Decay
Published Research
Uncovering the isoform-resolution kinetic landscape of nonsense-mediated mRNA decay with EZbakR
Methods: PacBio Iso-Seq | Published: 2025 | PMC11952489
Background
Nonsense-mediated mRNA decay (NMD) is a quality-control pathway that eliminates mRNAs with premature termination codons. While bulk-level NMD targets are known, isoform-level NMD dynamics — which specific splice variants are targeted and how rapidly they decay — remain poorly understood because short-read RNA-seq cannot resolve full-length isoforms.
Materials & Methods
Experimental Design
- Metabolic labeling (EZbakR)
- Time-course sampling
- Biological replicates
Sequencing
- PacBio Iso-Seq
- Full-length cDNA
- Kinnex concatenation
Analysis
- Isoform discovery
- Kinetic modeling
- Differential decay rates
Results
- Isoform-specific NMD targeting
- A single gene produces both NMD-sensitive and NMD-resistant isoforms depending on alternative splicing.
- Isoform-resolution kinetic modeling revealed a hierarchy of decay rates invisible to bulk RNA-seq methods.
- Kinetic landscape of NMD
- Transcript-specific decay rate quantification
- EZbakR + Iso-Seq enabled direct measurement of isoform half-lives.
- Identified NMD escape mechanisms at the isoform level that cannot be detected by gene-level analysis.
Conclusion
This study demonstrates that PacBio full-length Iso-Seq enables isoform-resolution kinetic studies impossible with short-read RNA-seq. For researchers studying alternative splicing, post-transcriptional regulation, or isoform-level gene expression, Iso-Seq provides the molecular resolution to ask mechanistic questions — and get answers at the level of individual transcript isoforms.
Reference
- Uncovering the isoform-resolution kinetic landscape of nonsense-mediated mRNA decay with EZbakR. PMC11952489, 2025. https://pmc.ncbi.nlm.nih.gov/articles/PMC11952489/
Related Publications
Here are publications from researchers who have used our PacBio SMRT sequencing services or related platforms:
The first high-quality genome assembly and annotation of Lantana camara, an important ornamental plant and a major invasive species
Journal: Horticulture Advances
Year: 2024
DOI: 10.1007/s44281-024-00043-6
Complete Genome Sequence of the Lignocellulose-Degrading Actinomycete Streptomyces albus CAS922
Journal: Microbiology Resource Announcements
Year: 2020
DOI: 10.1128/mra.00227-20
A chromosome-scale and haplotype-resolved genome assembly of tetraploid blackberry (Rubus L. subgenus Rubus)
Journal: Horticulture Research
Year: 2025
DOI: 10.1093/hr/uhaf052
See more articles published by our clients.
