PacBio Single Molecule Sequencing

GGBC is not currently offering PacBio Sequel II Sequencing

Please see the following link for PacBio service providers.  

General Information on Sequel II and Revio Sequencing Systems

The PacBio platform is based on Single Molecule, Real-Time (SMRT) technology. It is ideal for rapidly and cost-effectively generating high-quality whole genome de novo assemblies, full-length transcriptomes, cell type-specific isoforms (MAS-Seq) and long-read targeted amplicon sequences.

The new PacBio Revio system has all the capabilities of the Sequel II but with a higher throughput (up to 3X per SMRT-cell), better accuracy, and shorter run times for a lower cost per base than on the Sequel II.

At this time, Iso-Seq libraries and libraries for fragments shorter than 3 kb (e.g. short amplicons) need to be sequenced on the Sequel II. However, new library preparation kits for the Revio will be available shortly. Note that the yield per SMRT cell is dependent on original material source or organism and cannot be guaranteed.

Image courtesy of PacBio (Pacific Biosciences)

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:

  • Yaravi Zuleira Suarez (yzs96757@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

DNA Extraction Protocols

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

ApplicationLibrary TypeDescriptionMultiplexingMass RequirementVolume RequirementOther Requirements
Whole Genome SequencingDe Novo Assembly - HiFi ReadsProduce reference quality assemblies for genomes_1 ug/ Gb of genome≥50 uL50% ≥ 30 kb & 90% ≥ 10 kb
Variant DetectionWith 2 SMRT Cells 8M, Call SNVs, InDels, and SVs in a 3 Gb genome_1 ug/ Gb of genome≥50 uL50% ≥ 30 kb & 90% ≥ 10 kb
De Novo Assembly - for Low DNA InputProduce reference quality assemblies for genomes up to 1 Gb. Multiplex up to 2 small genomes on the Sequel II Systemtwo 600-Mb genomes>400 ng per 1 Gb genome size (single-sample), >300 ng per 600 mb genome size (2-plex)≥50 uL50% ≥ 30 kb & 90% ≥ 10 kb
Microbial De Novo AssemblySequence up to 96 microbes up to 96 microbes>300 ng per microbe≥50 uL90% ≥ 7 kb
De Novo Assembly and Variant Detection - for UltraLow DNA InputProduce reference quality assemblies for genomes up to 500 Mb_5-20 ng per 500 Mb genome size≥50 uLMajority of gDNA >20 kb
Viral SequencingHiFiViral SARS-CoV-2This procedure captures the SARSCoV2 genome with tiled molecular inversion probes that create overlapping amplicons resulting in comprehensive sequence coverageup 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 issuesup 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 SequencingIso-Seq MethodCharacterize alternative splicing/annotate a genome with full length transcriptsup to 12 Iso-Seq samples/SMRT Cell 8M for genome annotation300 ng total RNA≥10 uLRIN (RNA integrity number) ≥7.0
MAS-Seq for 10x Single CellIdentify 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 uLOptimal range of 3,000-10,000 target cell recovery from the 10x Chromium 3’ single cell workflow
MetagenomicsFull-length 16S rRNA SequencingMultiplex up to 192 samples to provide strain level resolutionup to 192 samples1000 ng of pooled 16S samples≥50 uL for a pool_
Shotgun Metagenomic Profiling or AssemblyGenerate 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 uL90% ≥ 7 kb
Targeted SequencingAmplicon SequencingHiFi sequencing can span your amplicon in a single read, giving unambiguous haplotype resolution through direct phasingup to 1,000+ samplesTotal 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 Table

PacBio ServiceDescriptionUGA FeeNon-UGA FeeCommercial Fee
HMW DNA or RNA
Extraction
DNA or RNA QC prior to extraction/purificationVolume, Concentration (ng/ul) and
DNA Size
InquireInquireInquire
HMW DNA extraction
from Plant leaf tissue
High molecular Weight DNA
extraction suitable for HiFi library
preparation
InquireInquireInquire
RNA preparationRNA preparation from frozen tissueInquireInquireInquire
Library Preparation
CCExpress Library (HiFi)
Indexed
10 µg HMW DNA minimum
required
InquireInquireInquire
IsoSeq ExpressExpress IsoSeq library sized with
beads
InquireInquireInquire
Sequencing
Revio 24-h movie timeIsoSeq and short amplicons not
currently supported
InquireInquireInquire
Sequel II 30-h movie timeIsoSeq and short amplicons are
supported
InquireInquireInquire
Data Analysis
Data AnalysisSee https://dna.uga.edu/bioinformaticsInquireInquireInquire

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.