PacBio Single Molecule Sequencing

Sequel II System: high-throughput, cost-effective access to SMRT Sequencing
The Sequel II System 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. At GGBC we provide all Sequel II based services, from library prep and sequencing to data analysis.
GGBC has sequenced a variety of different organisms including human, plant, animal, insect, bacteria, fungi, yeast, and others.

Image courtesy of PacBio (Pacific Biosciences)

Sequel II Instrument at the GGBC

Consultation and Assistance

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.
Library Preparation Types and Requirements

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_
Prices and Quotes

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

StepServiceUGA FeeNon-UGA FeeCommercial Fee
1. QCDNA quality assessment for PacBio library$16$18.90$20
RNA quality assessment for PacBio library (per group of 11 samples)$92$109$115
2. LibrariesFull-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 LibrariesMultiplexed 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 QCQuality assessment of final SMRTbell library/pool$63$74.50$78.75
4. SequencingDNA 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
Data Retrieval

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.

Applications