PacBio Pre-Made Library Sequencing — Submit Your SMRTbell Libraries for HiFi Sequencing
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PacBio Pre-Made Library Sequencing — Submit Your SMRTbell Libraries for HiFi Sequencing

PacBio HiFi sequencing of pre-made library on Revio

You have built the libraries. We run the sequencers. CD Genomics accepts pre-constructed SMRTbell libraries from labs worldwide for PacBio HiFi sequencing on both Sequel II/IIe and Revio platforms. Library QC is performed before every run so you never commit a SMRT Cell without knowing your library is ready. What you get back: demultiplexed CCS reads at Q30+ accuracy, complete run metrics, and optional bioinformatics support.

Why researchers choose our pre-made library sequencing service

At a glance:

Sequencing-Only PacBio HiFi — Submit Your Library, We Handle the Run

Research teams invest significant resources in building SMRTbell libraries — DNA extraction, shearing, size selection, adapter ligation, and QC. By the time the library is ready, the decision to sequence it in-house or outsource it becomes a calculation of instrument availability, queue length, and cost per SMRT Cell.

Our sequencing-only service is built for labs that already have library preparation workflows and need a reliable sequencing partner. You ship us your pre-made libraries on dry ice, we QC them, load them onto the appropriate SMRT Cell, and deliver HiFi sequencing data. No library preparation, no DNA extraction — just sequencing, QC, and data delivery.

Who this service is for

This service is designed for three groups: core facilities with PacBio library prep capability but insufficient sequencer capacity to meet demand, academic PIs whose labs prepare SMRTbell libraries but cannot justify a full instrument purchase, and biotech or agricultural genomics teams running internal library prep workflows at scale and needing consistent, high-quality sequencing output. In each case, the lab has already invested in library preparation; our role is to provide the sequencing capacity.

What we do with your library

When your library arrives, our first action is quality control — not sequencing. We measure concentration by Qubit fluorometry, assess fragment size distribution by Bioanalyzer or TapeStation, and verify SMRTbell adapter integrity. Only after the QC report confirms the library meets our loading criteria do we proceed to SMRT Cell loading and sequencing. This gate prevents wasted SMRT Cells and ensures the data you receive reflects your library quality, not instrument variability.

Key differentiators vs full-service sequencing

Most PacBio sequencing providers bundle library preparation with sequencing as a single service package. While this works for labs that prefer end-to-end outsourcing, it creates unnecessary cost and turnaround time for labs that prepare their own libraries. By separating library prep from sequencing, we deliver sequencing-only service with faster turnaround — because we skip the library preparation step entirely. For a comparison of the PacBio platforms available through our service, see our PacBio SMRT Sequencing Technology page. If you are also evaluating nanopore-based sequencing options, we offer a parallel Oxford Nanopore Pre-Made Library Sequencing service for labs that use both platforms.

Library Quality Control — Verify Before You Commit a Full SMRT Cell

The single most common concern from researchers submitting pre-made libraries is straightforward: "Will you check my library before running it?" The answer is yes — and we provide the QC report before loading, so you make the final decision.

QC workflow: concentration, size, and adapter verification

Library QC follows a three-step protocol. First, concentration is measured by Qubit fluorometry using the dsDNA HS assay kit, which specifically quantifies double-stranded SMRTbell library molecules without interference from free adapters or nucleotides. We require a minimum concentration of 10 ng/µL and a minimum volume of 10 µL. Second, fragment size distribution is assessed by Agilent Bioanalyzer (or TapeStation for high-throughput submissions) to confirm the expected insert size range and verify the absence of adapter dimers or short fragments that would reduce effective sequencing yield. Third, we inspect the SMRTbell adapter peak — a distinct signature in the Bioanalyzer trace at approximately 200–300 bp that confirms successful adapter ligation.

Pass/fail criteria and reporting

A library passes QC when it meets three criteria: (1) Qubit concentration ≥ 10 ng/µL, (2) Bioanalyzer trace shows the expected fragment size distribution with a clean SMRTbell adapter peak, and (3) no evidence of adapter dimers or excessive short fragments (< 500 bp for WGS libraries). The QC report includes the Qubit concentration value, the Bioanalyzer electropherogram with peak annotations, and a pass/fail recommendation. This report is shared with you before any SMRT Cell is loaded.

What happens if your library doesn't pass QC

If a library does not meet our QC thresholds, we do not load it. Instead, we send you the QC report with specific findings — for example, concentration below the minimum, unexpected fragment size distribution, or missing adapter peak — along with our recommendations. You then choose from three options: submit a new library aliquot from the same preparation, adjust your library preparation protocol and resubmit, or cancel the sequencing run. You are not charged for a SMRT Cell that is never loaded.

PacBio Platform Options — Sequel II/IIe and Revio Systems

Choosing the right platform for your pre-made libraries depends on project scale, desired throughput, and budget. We maintain both Sequel II/IIe and Revio systems, each with distinct characteristics that make them suitable for different project types.

Feature Sequel II/IIe Revio
SMRT Cell Type SMRT Cell 8M (8 million ZMWs) SMRT Cell 25M (25 million ZMWs)
HiFi Yield per Cell 30–50 Gb 90–120 Gb
HiFi Reads per Cell ~1.5 million ~4 million
Read Length N50 15–25 kb 15–25 kb
Consensus Accuracy >99.9% (Q30+) >99.9% (Q30+)
Run Time per Cell ~30 hours ~24 hours
Cells per Day 1 (single-stage) 4 (4-stage parallel)
Daily Throughput 30–50 Gb/day 360–480 Gb/day
Best For Small-to-medium projects, cost-sensitive Large-scale projects, high-throughput

Sequel II/IIe — SMRT Cell 8M

The Sequel II and IIe systems use the SMRT Cell 8M, which contains 8 million zero-mode waveguides (ZMWs). Each SMRT Cell 8M typically yields 30–50 Gb of HiFi data with read length N50 of 15–25 kb, depending on library insert size. The Sequel IIe adds on-instrument CCS analysis, generating HiFi reads directly during the run rather than requiring post-run computation. Sequel II/IIe is well-suited for small-to-medium bacterial genomes, targeted amplicon panels, individual human genome sequencing, and projects where cost per sample is more important than total throughput. If you are preparing libraries for Human Whole Genome Sequencing, Sequel II/IIe provides a cost-effective entry point with HiFi data quality equivalent to Revio.

Revio — SMRT Cell 25M

The Revio system represents a generational leap in throughput. Each SMRT Cell 25M contains 25 million ZMWs and yields 90–120 Gb of HiFi data — approximately three times the output of a Sequel II/IIe run. Revio runs four SMRT Cells in parallel with staggered loading, enabling a daily throughput of 360–480 Gb. Read length N50 remains in the 15–25 kb range, but the higher read count per cell (~4 million HiFi reads vs ~1.5 million on Sequel II) provides deeper coverage for large genomes and better statistical power for structural variant detection. Revio is the recommended platform for population-scale human genome projects, large plant and animal genomes, metagenomic studies requiring deep coverage, and any project where turnaround speed is critical.

How to choose: sequencing scale vs cost

The decision between Sequel II/IIe and Revio comes down to project scale and budget. For a single 3 Gb human genome at 30× coverage, one Sequel II/IIe SMRT Cell provides sufficient data (~90 Gb needed, covered by 2–3 cells); for a cohort of 100 genomes at 30× coverage, Revio's 4× daily throughput makes it the practical choice. Our team can help you select the right platform based on your genome size, coverage target, and project timeline. If you need integrated analysis of your sequencing data after the run, our PacBio Sequencing Data Analysis service provides genome assembly, variant calling, and annotation as optional add-ons.

Accepted Library Types — WGS, Amplicon, Iso-Seq, and More

We accept SMRTbell libraries prepared with standard PacBio library preparation kits (SMRTbell prep kit 3.0 and compatible earlier versions) across a wide range of library types. This section describes what we accept, any type-specific requirements, and which CD Genomics services connect to each library type.

Whole-genome and metagenomic libraries

We accept WGS libraries with insert sizes from approximately 10 kb to 30 kb, which covers the standard range for PacBio HiFi genome sequencing. Metagenomic WGS libraries prepared from environmental DNA, gut microbiome, or other complex community samples are also accepted, provided the library shows a clean Bioanalyzer trace without excessive adapter dimers. For metagenomic libraries, we recommend a minimum insert size of 5 kb to maximize read alignment specificity in complex mixtures.

Amplicon and targeted sequencing libraries

Amplicon libraries — including full-length 16S rRNA, ITS, and custom targeted amplicon panels — are fully supported. Because amplicon libraries have a narrow size distribution centered on the amplicon length, we pay particular attention to the Bioanalyzer trace during QC to confirm the expected peak and rule out non-specific amplification products. For Full-Length 16S/18S/ITS Amplicon Sequencing, we accept libraries with amplicon sizes from 1.5 kb to 5 kb on both Sequel II/IIe and Revio.

Iso-Seq and full-length transcript libraries

Iso-Seq (full-length isoform sequencing) libraries prepared with the SMRTbell prep kit 3.0 are accepted. Because Iso-Seq libraries require additional QC steps — specifically, verification of full-length cDNA enrichment — we recommend submitting Iso-Seq libraries at a higher concentration (15–20 ng/µL) to provide sufficient material for both QC and SMRT Cell loading. Our Full-Length Transcript Sequencing (Iso-Seq) service page provides additional details on Iso-Seq applications and data analysis.

Multiplexed and barcoded libraries

Barcoded multiplex libraries using standard PacBio barcoded adapter systems are fully supported. We demultiplex pooled libraries as part of standard data delivery. When submitting multiplexed libraries, include a barcode-to-sample mapping file (CSV or Excel format) with sample names, barcode sequences or IDs, and any relevant metadata. For custom or non-standard barcoding schemes, please contact our team before submission to confirm compatibility with our demultiplexing pipeline.

Sequencing Metrics and Quality Guarantees

When you submit a pre-made library, you need to know what data quality you can expect — and what happens if the run does not meet expectations. This section presents the typical sequencing performance metrics we achieve and our run quality guarantee.

HiFi yield expectations by platform

HiFi sequencing performance depends on both the platform and the library quality. On the Sequel II/IIe platform with a SMRT Cell 8M, a well-prepared WGS library with insert size of 15–20 kb typically yields 30–50 Gb of HiFi data with approximately 1.5 million HiFi reads. On the Revio platform with a SMRT Cell 25M, the same library typically yields 90–120 Gb of HiFi data with approximately 4 million HiFi reads. These yields assume libraries that pass our QC thresholds. Libraries with shorter insert sizes may produce higher read counts but lower total yield (Gb), as more ZMWs load successfully but generate shorter polymerase reads.

Accuracy and read length distributions

All HiFi data we deliver meets or exceeds Q30 accuracy (>99.9% consensus base accuracy), which is the standard for PacBio HiFi sequencing. Read length N50 typically falls in the 15–25 kb range for WGS libraries, driven primarily by the insert size of the submitted library rather than the sequencing chemistry. For amplicon libraries, read lengths correspond to the amplicon size. For Iso-Seq libraries, read lengths reflect the full-length cDNA population. Each delivery includes a run metrics report with read length distribution histograms, Q-score distribution, and per-ZMW loading efficiency data, so you can independently verify data quality.

Run quality guarantee — what if metrics fall short

We guarantee minimum HiFi yield thresholds: 25 Gb per SMRT Cell for Sequel II/IIe and 75 Gb per SMRT Cell for Revio. If a sequencing run falls below these thresholds due to instrument or chemistry issues — not due to library quality problems identified during QC — we re-run the library on a fresh SMRT Cell at no additional cost. This guarantee covers the sequencing output, not the biological content of the data, which depends on the submitted library. If the library passed QC and the run underperformed, we absorb the cost and re-run.

Service Workflow — Submit, QC, Sequence, Deliver

The end-to-end workflow from library submission to data delivery is designed to be transparent at every step. You know the status of your library before any irreversible step is taken.

Step 1: Library submission and metadata

Ship your pre-made SMRTbell libraries on dry ice in sealed tubes or plates with clear labeling. Include a completed metadata sheet with: sample names, barcode-to-sample assignments (for multiplexed libraries), expected insert size range, library type (WGS, amplicon, Iso-Seq, etc.), and any special handling instructions. For international shipments, we provide customs documentation guidance. Upon receipt, we log the shipment, document the condition of dry ice and sample containers, and store libraries at -20°C.

Step 2: Library QC — Bioanalyzer and Qubit

We aliquot 1–2 µL of each library for QC. Concentration is measured by Qubit fluorometry, fragment size distribution by Agilent Bioanalyzer or TapeStation, and the SMRTbell adapter peak is verified. Results are compiled into a QC report and shared with you. You confirm whether to proceed with sequencing. If the library passes, we move to SMRT Cell loading. If not, you choose to resubmit or cancel (no charge for libraries that do not proceed to loading).

Step 3: Sequencing run — SMRT Cell loading

Libraries that pass QC are loaded onto the appropriate SMRT Cell (8M for Sequel II/IIe, 25M for Revio) according to the binding and loading protocol for the SMRTbell prep kit version used. We monitor loading efficiency and polymerase read length distribution during the run and can provide real-time status updates on request.

Step 4: CCS generation and data QC

After the sequencing run completes, raw subread data is processed through the CCS (circular consensus sequencing) algorithm to generate HiFi reads. For Sequel II/IIe, CCS is performed post-run; for Revio, CCS is generated on-instrument. Post-CCS data QC includes: HiFi read count, total HiFi yield (Gb), read length N50, mean Q-score, and per-barcode read distribution (for multiplexed libraries). These metrics are compiled into the run QC report.

Step 5: Secure data delivery

Data is delivered via secure FTP or cloud storage (customer preference). Standard deliverables include: demultiplexed HiFi reads in FASTQ or BAM format, the run metrics report (PDF), and the initial library QC report. If you have specific data delivery requirements — such as direct upload to your institutional HPC or cloud bucket — we accommodate these on request.

PacBio pre-made library sequencing workflow from submission to data deliveryEnd-to-end service workflow: library submission → QC verification → SMRT Cell sequencing → CCS generation → secure data delivery.

Data Delivery and Bioinformatics Support

The data you receive from a sequencing run is the foundation for downstream analysis. We deliver standard sequencing outputs plus optional bioinformatics services for teams that need analysis support.

Standard data deliverables

Every sequencing run includes the following standard deliverables:

Run metrics and QC reporting

The run metrics report is designed to give you full transparency into sequencing performance. The Q-score distribution plot shows the proportion of bases at each quality score; HiFi reads should predominantly fall at Q30 and above. The read length distribution histogram helps you verify that the sequencing output matches your expected insert size. If you notice deviations — for example, a shorter-than-expected N50 — this information helps diagnose whether the issue originates in library preparation or sequencing.

Optional bioinformatics analysis add-ons

For teams that need analysis support beyond raw data delivery, we offer optional bioinformatics services:

All bioinformatics add-ons are performed by our dedicated analysis team with standardized pipelines and QC reporting. If you need custom analysis workflows beyond these standard offerings, please discuss your requirements with our team. For more information on our analysis capabilities, see the PacBio Sequencing Data Analysis page for full details on genome assembly, variant calling, and annotation support.

Library Shipping and Handling Requirements

Proper shipping is critical for library integrity. This section covers packaging requirements, minimum sample specifications, and documentation for international shipments.

Packaging and shipping on dry ice

SMRTbell libraries should be shipped frozen on dry ice in an insulated shipping container. For domestic shipments, 5–10 kg of dry ice is typically sufficient to maintain temperature for 24–48 hours. For international shipments, use 10–15 kg of dry ice and choose express shipping (2–3 day delivery) to minimize time in transit. We recommend including a temperature logger inside the shipping container to verify that the library remained at -20°C or below throughout transit. Upon receipt, we immediately document the condition of the dry ice and sample integrity, and transfer libraries to -20°C storage.

Minimum volume and concentration requirements

Each library submission must meet the following minimum specifications:

  • Volume: ≥ 10 µL per library (single-library submissions) or ≥ 5 µL per library (multiplexed pools, where the pool represents a single sample for QC purposes).
  • Concentration: ≥ 10 ng/µL as measured by Qubit dsDNA HS assay. For Iso-Seq libraries, we recommend ≥ 15 ng/µL to account for additional QC steps.
  • Container: Sealed microcentrifuge tubes (1.5 mL or 0.5 mL) or sealed 96-well plates with pierceable seals. Label each tube or plate with sample name and date.
  • Buffer: Libraries should be in EB (10 mM Tris-Cl, pH 8.5) or equivalent low-EDTA buffer. Avoid buffers with high EDTA concentrations (> 1 mM), as EDTA can interfere with SMRT Cell loading chemistry.

Metadata and documentation checklist

Include the following with every shipment:

  1. Sample manifest: CSV or Excel file with sample names, barcode sequences/IDs (for multiplexed libraries), library type, expected insert size range, and any special notes.
  2. Barcode-to-sample mapping: Required for multiplexed libraries. Include barcode ID or sequence and corresponding sample name.
  3. Shipping declaration: For international shipments, a customs declaration form listing "non-hazardous, non-infectious DNA library for research purposes" is typically required. Contact our team for destination-specific customs guidance.
  4. Contact information: Name, email, and phone number of the person to contact with QC results and questions.

Case Study — HiFi Data Quality Enables Paralogous Gene Resolution

The quality of HiFi sequencing data is not an abstract specification — it determines what biological questions you can answer. This case study, drawn from published research, illustrates how the data quality our service delivers enables analysis that is impossible with lower-quality sequencing approaches.

Background: the paralogous gene challenge

Paralogous genes in segmental duplications share >99% sequence identity over regions exceeding 10 kb. Short-read sequencing cannot resolve these regions because reads shorter than the duplicated segment cannot be uniquely mapped — they align equally well to multiple genomic locations. This creates systematic blind spots in human genome analysis, precisely in regions that contain medically critical genes including CYP21A2 (congenital adrenal hyperplasia), SMN1/SMN2 (spinal muscular atrophy), and HBA1/HBA2 (alpha-thalassemia). Resolving these genes requires long, accurate reads that span entire duplicated segments.

Methods: Paraphase pipeline and PacBio HiFi CCS

Chen et al. (2025) developed Paraphase, a computational pipeline that uses PacBio HiFi CCS reads to genotype paralogous genes genome-wide. The study sequenced and analyzed 160 paralogous regions — each >10 kb with >99% sequence identity — across 259 individuals from five ancestral populations (African, Admixed American, East Asian, European, and South Asian). Libraries were prepared with standard SMRTbell protocols and sequenced on PacBio systems to generate HiFi reads with Q30+ accuracy — the same data quality our service delivers.

Results: 316 genes, 23 low-diversity groups, 11 de novo events

The Paraphase pipeline successfully genotyped 316 genes across segmental duplications, identifying 23 low-diversity paralog groups with minimal haplotype variation across populations. The analysis detected 7 de novo single-nucleotide variants and 4 de novo gene conversion events — including 2 non-allelic gene conversions — that were invisible to short-read approaches. Nine medically relevant genes were fully resolved, demonstrating that PacBio HiFi data quality is the prerequisite for accurate analysis of structurally complex genomic regions.

Conclusions: HiFi data quality is the prerequisite for complex genomic analysis

This study demonstrates what becomes possible when sequencing data quality is sufficient to span entire segmental duplications with high accuracy. The HiFi reads used in this analysis — Q30+, 15–25 kb, generated from standard SMRTbell libraries — represent exactly the data type our sequencing-only service delivers. For researchers working on medically relevant paralogous genes, structural variant discovery, or any project involving complex genomic regions, the quality of the sequencing data determines the quality of the conclusions. Source: Genome-wide profiling of highly similar paralogous genes using HiFi sequencing, Chen X et al., Nature Communications, 2025.

Paraphase pipeline for paralogous gene resolution using PacBio HiFi sequencing dataCase study figure: Paraphase pipeline workflow for paralogous gene resolution using PacBio HiFi CCS reads (left) and comparison of HiFi vs Illumina read mapping quality at a representative paralogous locus (right). Adapted from Chen et al. 2025, Nature Communications (CC BY 4.0).

Demo Results — Sequencing Performance Data

The following representative data illustrate the sequencing performance customers can expect when submitting pre-made libraries for PacBio HiFi sequencing on our platforms.

Typical HiFi yield by library type and platform

Library Type Sequel II/IIe (Gb/SMRT Cell) Revio (Gb/SMRT Cell) Typical Read Count (Revio)
WGS (human, 15 kb insert) 35–45 100–120 3.5–4.5 million
WGS (bacterial, 10 kb insert) 40–55 110–130 4.0–5.0 million
Metagenomic WGS (5–10 kb) 30–40 90–110 3.0–4.0 million
Amplicon (16S full-length) 45–55 120–140 4.5–5.5 million
Iso-Seq (full-length cDNA) 25–35 80–100 2.5–3.5 million
Targeted capture 35–45 100–115 3.5–4.0 million

These values represent typical performance for libraries that pass our QC thresholds. Actual yield depends on library insert size, loading efficiency, and polymerase performance. The Revio platform consistently delivers approximately 3× the HiFi yield of Sequel II/IIe per SMRT Cell, consistent with the 3× increase in ZMW count from SMRT Cell 8M to 25M.

Representative read length distribution

For a typical WGS library with ~15 kb insert size, the HiFi read length distribution shows a mode at approximately 15 kb and an N50 of 18–22 kb, reflecting the combined effect of insert size selection and polymerase processivity. Read lengths above 30 kb are observed in the tail of the distribution, and reads below 5 kb represent a small minority originating from shorter library fragments. For amplicon libraries, the read length distribution is tightly centered on the amplicon size with minimal dispersion. Each delivery includes the full read length distribution histogram in the run metrics report.

Quality score distribution

Across all library types and both platforms, >90% of HiFi bases achieve Q30 or higher, and the mean Q-score typically exceeds Q33. The Q-score distribution is generated per SMRT Cell and included in every run metrics report, providing transparent documentation of sequencing accuracy.

PacBio HiFi sequencing demo results showing yield comparison across library types and platformsDemo results: representative HiFi yield (Gb per SMRT Cell) by library type on Sequel II/IIe and Revio platforms. Dashed reference line indicates Revio minimum quality guarantee threshold.

References

  1. Long-read human genome sequencing and its applications. Logsdon GA, Vollger MR, Eichler EE. Nature Reviews Genetics, 2020, 21(10): 597–614.
  2. Genome-wide profiling of highly similar paralogous genes using HiFi sequencing. Chen X, Baker D, Dolzhenko E, et al. Nature Communications, 2025, 16: 2340.
  3. Long and Accurate: How HiFi Sequencing is Transforming Genomics. Wang Y, Zhao Y, Bollas A, Wang Y, Au KF. Genomics, Proteomics & Bioinformatics, 2025, 23(1): qzaf003.

Frequently Asked Questions

Ready to Submit Your Libraries?

If you have SMRTbell libraries ready for sequencing, our team can provide a project-specific quote and confirm platform availability for your timeline. Tell us about your libraries — type, quantity, expected insert size, and preferred platform — and we will respond with a sequencing plan and next steps for submission.

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Disclaimer: CD Genomics provides PacBio HiFi sequencing services for research use only (RUO). Data and analysis results generated through our services are intended for research purposes and should not be used for clinical diagnosis, treatment decisions, or any patient management without independent validation. All services are performed in accordance with our standard operating procedures and quality management system.

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