PacBio Single Molecule Sequencing
Image courtesy of PacBio (Pacific Biosciences)
Sequel II Instrument at the GGBC
Contact for Genomics and Bioinformatics Consultation
Details about sample preparation, requirements, library prep types, prices and application are all listed on this page. For more information and assistance:
- Magdy Alabady (malabady@uga.edu) – consultation on new or existing Genomics and Bioinformatics projects, assistance with grant proposals and obtaining a letter of support from GGBC
- Kateryna Stoianova (kstoianova@uga.edu), Genomics Professional, for technical questions about existing or new PacBio projects.
- Kaitlyn Camp (kaitlyn.camp@uga.edu), Genomics Specialist, for technical questions about existing or new PacBio projects.
- Please visit our “All Inquiries” page for detailed information about who you should contact at GGBC to receive a quick and accurate response.
Helpful Links Related to PacBio Sample Preparation
PacBio Nanobind HMW DNA extraction overview
Technical-Note-Preparing-DNA-for-PacBio-HiFi-Sequencing-Extraction-and-Quality-Control
High-quality, high-molecular-weight genomic DNA is crucial for obtaining long read lengths and optimal sequencing performance. The SMRT library preparation process does not utilize amplification techniques and resulting library molecules are used directly as templates for the sequencing process. The quality of the DNA and RNA starting material will directly impact the extent of sequencing success or failure. Any unrepaired or irreversible DNA damage present in the input material (e.g., interstrand crosslinks, nicks, etc.) or contaminants will result in impaired performance in the system.
Upon receiving your DNA sample, a QC of Qubit, Nanodrop, and Fragment Analyzer is performed to ensure the DNA meets our concentration, purity, and size requirements, respectively.
Ideally, the 260/230 should be within the 2.0-2.2 range, and the 260/280 within 1.8-2.0 range. Initial DNA/RNA quality is critical for a successful run.
If you are submitting 24 or more samples, please submit samples in a 96-well plate and include an Excel file with the sample layout in your order.
PLEASE USE V BOTTOM PLATES DESIGNED FOR MOLECULAR BIOLOGY AND NOT FLAT BOTTOM PLATES DESIGNED FOR OTHER PURPOSES.
If your samples do not meet the requirements in the table below, please contact us to discuss potential options for processing your project.
PacBio Library Prep
| Application | Library Type | Description | Multiplexing | Mass Requirement | Volume Requirement | Other Requirements |
|---|---|---|---|---|---|---|
| Whole Genome Sequencing | De Novo Assembly - HiFi Reads | Produce reference quality assemblies for genomes | _ | 1 ug/ Gb of genome | ≥50 uL | 50% ≥ 30 kb & 90% ≥ 10 kb |
| Variant Detection | With 2 SMRT Cells 8M, Call SNVs, InDels, and SVs in a 3 Gb genome | _ | 1 ug/ Gb of genome | ≥50 uL | 50% ≥ 30 kb & 90% ≥ 10 kb | |
| De Novo Assembly - for Low DNA Input | Produce reference quality assemblies for genomes up to 1 Gb. Multiplex up to 2 small genomes on the Sequel II System | two 600-Mb genomes | >400 ng per 1 Gb genome size (single-sample), >300 ng per 600 mb genome size (2-plex) | ≥50 uL | 50% ≥ 30 kb & 90% ≥ 10 kb | |
| Microbial De Novo Assembly | Sequence up to 96 microbes | up to 96 microbes | >300 ng per microbe | ≥50 uL | 90% ≥ 7 kb | |
| De Novo Assembly and Variant Detection - for UltraLow DNA Input | Produce reference quality assemblies for genomes up to 500 Mb | _ | 5-20 ng per 500 Mb genome size | ≥50 uL | Majority of gDNA >20 kb | |
| Viral Sequencing | HiFiViral SARS-CoV-2 | This procedure captures the SARSCoV2 genome with tiled molecular inversion probes that create overlapping amplicons resulting in comprehensive sequence coverage | up to 384 SARS-CoV-2 samples | >10,000 copies of SARS-CoV-2 RNA per sample | ≥10 uL | _ |
| Adeno-associated virus (AAV) | Generate long and accurateHiFi reads, which can sequence an AAV genome from ITR to ITR, revealing otherwise difficult-to-detect issues | up to 24 AAV samples | ≥ 1 µg of AAV DNA per SMRT Cell, or per sample amounts should be 1 µg/ number of samples | ≥50 uL | _ | |
| RNA Sequencing | Iso-Seq Method | Characterize alternative splicing/annotate a genome with full length transcripts | up to 12 Iso-Seq samples/SMRT Cell 8M for genome annotation | 300 ng total RNA | ≥10 uL | RIN (RNA integrity number) ≥7.0 |
| MAS-Seq for 10x Single Cell | Identify cell type-specific isoforms and take advantage of long and accurate HiFi read lengths to increase single-cell Iso-Seq throughput by 16-fold through concatenated arrays of single-cell cDNA molecules | _ | 15 ng per library or 60-75 ng per library | ≥15 uL | Optimal range of 3,000-10,000 target cell recovery from the 10x Chromium 3’ single cell workflow | |
| Metagenomics | Full-length 16S rRNA Sequencing | Multiplex up to 192 samples to provide strain level resolution | up to 192 samples | 1000 ng of pooled 16S samples | ≥50 uL for a pool | _ |
| Shotgun Metagenomic Profiling or Assembly | Generate near-complette assemblies of high-complexity sample(s) (e.g. gut microbiome) | profile up to 48 communities, assemble up to 4 communities | >300 ng per sample | ≥50 uL | 90% ≥ 7 kb | |
| Targeted Sequencing | Amplicon Sequencing | HiFi sequencing can span your amplicon in a single read, giving unambiguous haplotype resolution through direct phasing | up to 1,000+ samples | Total input DNA per SMRT Cell 8M: 300 ng for <3 kb, 500 ng for 3 - 10 kb, ≥1000 ng for ≥10 kb | ≥50 uL for a pool | _ |
Contact for Financial Inquiries and Quote Requests
Please email Kim and Elizabeth at ggbc@uga.edu, for financial inquiries or to request a quote. Be as specific as possible, so that they can more quickly assist you.
A Note About Single SMRTcell Projects:
SMRTcell sequencing results depend on the final loading concentration of the library. Due to the nature of the technology, optimal concentrations can vary greatly between libraries and PacBio recommends running 2 titration SMRTcells to find the optimal concentration before running production SMRTcells. With this in mind, please be aware that projects requesting a single SMRTcell may have variable results. In most cases the first SMRTcell performs well and this is not an issue, but sometimes additional sequencing is needed. When this happens, the customer is expected to cover the cost of additional sequencing.
PacBio Sequel II DNA library preparation and SMRT cell run fees
| Step | Service | UGA Fee | Non-UGA Fee | Commercial Fee |
|---|---|---|---|---|
| 1. QC | DNA quality assessment for PacBio library | $16 | $18.90 | $20 |
| RNA quality assessment for PacBio library (per group of 11 samples) | $92 | $109 | $115 | |
| 2. Libraries | Full-length Barcoded Amplicons of 16S/18S/ITS (1-24 samples) | $1,345 | $1,588 | $1,682 |
| Full-length Barcoded Amplicons of 16S/18S/ITS (25-48 samples) | $1,617 | $1,909 | $2,022 | |
| Full-length Barcoded Amplicons of 16S/18S/ITS (49-72 samples) | $1,940 | $2,290 | $2,425 | |
| Full-length Barcoded Amplicons of 16S/18S/ITS (73-96 samples) | $2,259 | $2,666 | $2,824 | |
| Library from Amplicons | $617 | $729 | $772 | |
| Ultra Low input (non multiplexed) HiFi library | $1,235 | $1,458 | $1,544 | |
| High Fidelity Library (HiFi) (non multiplexed) | $655 | $773 | $819 | |
| Large SMRTbell library (15-20kb and >30kb)(can be multiplexed; see price below) | $533 | $629 | $667 | |
| IsoSeq Library | $584 | $690 | $730 | |
| MAS-Seq for 10xSingle Cell 3' Concatenation | $1,363 | $1,609 | $1,704 | |
| HiFi Viral library (per 96-well plate) | $5,744 | $6,778 | $7,180 | |
| xGen Hybridization using IDT probes | $900 | $1,062 | $1,125 | |
| 2. Multiplexed Libraries | Multiplexed SMRTbell genomic libraries: Pre-multiplexing (per sample) | $399 | $471 | $499 |
| Multiplexed SMRTbell genomic libraries: Post-multiplexing (per pool) | $143 | $169 | $179 | |
| Pre-multiplexing PCR Barcoded IsoSeq libraries (per sample) | $352 | $416 | $440 | |
| Post-multiplexing PCR Barcoded IsoSeq libraries (per pool) | $352 | $416 | $440 | |
| Pre-multiplexing Ligation Barcoded IsoSeq libraries (per sample) | $545 | $644 | $682 | |
| Post-multiplexing Ligation Barcoded IsoSeq libraries (per pool) | $30 | $35.40 | $37.50 | |
| 3. Library QC | Quality assessment of final SMRTbell library/pool | $63 | $74.50 | $78.75 |
| 4. Sequencing | DNA library binding, annealing, and cleanup prior to sequencing | $285 | $337 | $357 |
| PacBio Sequel SMRTCell sequencing run for CLR/HiFi (15 hrs) | $1774 | $2094 | $2218 | |
| PacBio Sequel SMRTCell sequencing run for IsoSeq (26 hrs) | $2100 | $2478 | $2625 | |
| PacBio Sequel SMRTCell sequencing run for HiFi (30 hrs) | $2400 | $2832 | $3000 | |
The standard output files from the PacBio sequencing run are:
| Raw subreads | CCS (HiFi) reads |
| *.baz2bam_1.log | ccs_processing.report.json |
| *.scraps.bam | ccs.report.csv.zip |
| *.scraps.bam.pbi | ccs.report.json |
| *.sts.xml | ccs_tasks_report.json |
| *.subreads.bam | ccs_zmws.json.gz |
| *.subreads.bam.pbi | final.consensusreadset.xml |
| *.subreadset.xml | *.hifi_reads.bam |
| *.transferdone | *.hifi_reads.fasta.gz |
| tmp-file*.txt | *.hifi_reads.fastq.gz |
| reads.bam |
We will be sending you the following files:
| Raw subreads | CCS (HiFi) reads |
| *.baz2bam_1.log | ccs.report.csv.zip |
| *.sts.xml | final.consensusreadset.xml |
| *.subreads.bam | *.hifi_reads.bam |
| *.subreads.bam.pbi | *.hifi_reads.fasta.gz |
| *.hifi_reads.fastq.gz | |
| reads.bam |
For multiplexed runs, you will be receiving both raw and demultiplexed data.
GGBC uses Globus to transfer the data. We ask that you please confirm when you received and downloaded the data. Once your download link expires, your data is deleted on our end so please contact us beforehand if you have issues downloading the data.
- Whole Genome Sequencing:
- Targeted Sequencing:
- RNA Sequencing:
- Epigenetics Sequencing:
- Metagenome Sequencing
