PacBio Single Molecule Sequencing

Sequel II System: high-throughput, cost-effective access to SMRT Sequencing                                                                                          

The Sequel System is based on Single Molecule, Real-Time (SMRT) technology and is ideal for rapidly and cost-effectively generating high-quality PacBio whole genome de novo assemblies and full-length transcriptomes. At GGBC, we provide all Sequel based services, from library prep and sequencing to data analysis.

PacBio Sequel II sequencing modes:

CLR (Continuous Long Read)

With reads tens of kilobases in length you can readily assemble complete genomes and sequence full-length transcripts. SMRT Sequencing provides exceptional read lengths without compromising throughput or accuracy. Traditional PacBio long reads are generated by a single pass of DNA Polymerase around a circularized template, which allows for overall longer read lengths but does not provide the higher accuracy characteristic of HiFi reads.

CCS (Circular Consensus Sequences)

Using SMRT technology, highly accurate long reads known as High Fidelity reads (HiFi) are attainable with an accuracy of >99.9%. This method ensures both long read sequencing depth, and high accuracy. HiFi reads are the data produced using CCS. During library preparation, SMRTbell adapters are ligated to double-stranded DNA, thus circularizing the library. Primers and DNA polymerase are annealed and the circularized library is sequenced in repeated passes of the enzyme around the template. Consensus is called from the multiple subreads to provide a highly accurate long HiFi read.  Click here for more information on CCS.

More Information: Overview-Sequel-Systems-Application-Options-and-Sequencing-Recommendations

Please see the Application section below for more information.

About Sequel II:

  • Generates ~8-times more data than the original Sequel System     (80Gbp +/- 20%)
  • Provides access to even more highly accurate long reads (HiFi Reads)
  • Reduces project time for faster results
  • Makes sequencing more affordable
  • Supports the range of SMRT Sequencing applications

Webinar: Sequence with Confidence – Introducing the Sequel II System

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), Lab Technician, 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

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.  Samples will then undergo a back-to-back bead cleanup using AMPure PB beads to ensure DNA is as clean as possible to begin library prep. Due to this process, we may require additional material. The percentage loss can vary depending on the purity of your initial DNA submission.

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.

The table below shows our most requested library preparation types.  For more information and other library types, see the following link: https://www.pacb.com/wp-content/uploads/Overview-Sequel-Systems-Application-Options-and-Sequencing-Recommendations.pdf

Application HiFiLow Input- HiFi Ultra-Low InputHiFi MultiplexIso-Seq Large Insert
Required Amount7 ug>600 ng per 1 kb genome >400 ng per 600 Mb genome (2-plex)5-20 ng per 500 Mb genome size2 ug per sample300 ng per sample4-7 up per sample
Required Volume50-200 uL50-200 uL2-50 uL50-200 uL≥40 ng/uL in minimum of 15 uL50-200 uL
Size> 40 kb> 30 kb> 20 kb> 20 kbA RIN ≥7.0 (ideally ≥8.0) is sufficient. Samples with a RIN <7.0 can be processed, but the risk of significant underperformance or even failure is greatly increased > 50 kb
Multiplex AvailableNoNoNoUp to 48 microbesUp to 12No
Data TypeHiFi / CCSHiFi / CCSHiFi / CCSCLR / CCS*CSSCLR
Run Time30 hr.30 hr.30 hr.15 hr.24 hr.15 hr.

*Can run as HiFi to produce CCS data, if desired

Other Helpful Links Related to PacBio Sample Preparation

PacBio Guidelines for Successful SMRTbell Libraries
Q&A for “DNA Quality Requirements for Single Molecule, Real-Time (SMRT) Sequencing”
Note on the 260/280 and 260/230 Ratios
DNA Extraction Protocols: www.ExtractDNAforPacBio.com

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$37$43.70$46.25
RNA quality assessment for PacBio library (per group of 11 samples)$86$102$108
2. LibrariesFull-length Barcoded Amplicons of custom/16S/18S/ITS (1-48 samples)$797$941$997
Full-length Barcoded Amplicons of custom/16S/18S/ITS (49 -96 samples)$1159$1368$1449
Library from Amplicons$533$629$667
Low input (non multiplexed) HiFi library$533$629$667
Ultra Low input (non multiplexed) HiFi library$1189$1404$1487
High Fidelity Library (HiFi; includes SageELF run)(non multiplexed)$713$842$892
Large SMRTbell library (15-20kb and >30kb)(can be multiplexed; see price below)$533$629$667
IsoSeq Library$627$740$784
xGen Hybridization using IDT probes$748$883$935
2. Multiplexed LibrariesMutliplexed SMRTbell genomic libraries: Pre-multiplexing (per sample)$216$255$270
Mutliplexed SMRTbell genomic libraries: Post-multiplexing (per pool)$166$196$208
Multiplexed Isoseq libraies: Pre-multiplexing (per sample)$424$501$530
Multiplexing Isoseq libraries: Post-multiplexing (per pool)$204$241$255
3. Library QCQuality assessment of final SMRTbell library/pool$29$34.25$36.25
4. SequencingDNA library binding, annealing, and cleanup prior to sequencing$230$272$288
PacBio Sequel SMRTCell sequencing run for CLR (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:

  • *.log
  • *.scraps.bam
  • *.scraps.bam.pbi
  • *.sts.xml
  • *.subreads.bam
  • *.subreads.bam.pbi
  • *.subreadset.xml
  • *.transferdone
  • *tmp-file-*.txt

We will be sending you the following files:

  • *.log
  • *.sts.xml
  • *.subreads.bam
  • *.subreads.bam.pbi

For multiplexed runs, you will be receiving both raw and demultiplexed data.

CCS data can be generated per request and will require a separate bioinformatics order.

Data files will be transferred to customers through the GGBC’s data distribution FEX server. The FEX download links will be active for up to 2 weeks from the time you receive them. Please make sure to have enough time and storage for downloading your data.

We also 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.

Sequenced Species

Table 1. Organisms Sequenced on the PacBio Sequel at GGBC

IsoSeq 
AzaleaRhododendron canescens
CopepodsNeocalanus flemingeri, Pseudocalanus, Metridia pacifica
FishGasterosteus aculeatus, Polyodon spathula
GerbilMongolian gerbil
MosquitoAnopheles stehpensi
NematodeBipalium kewense
PlantBipalium kewense, Red Palm Weevil
Synthetic RNA
Large Molecule Library
AlgaePicocystis salinarum
AnimalAnolis sagrei, Heloderma charlesbogerti, Homarus americanus, Stickleback fish
BacteriaAetokthonos hydrillicola, Bacillus, Bifidobacterium, Delftia, Rhodococcus, Serratia, Sphingobacterium, Xylella fastidiosa
FungiGanoderma, Hymenochaete, Meyerozyma, Oudemansiella, Phlebia, Pleurotus flabellatu, Trametes cubensis, Trametes elegans
InsectAdelges cooleyi, Aphid, moth, Osmia lignaria
NematodeCaenorhabditis elegans, Dirofilaria immitis
PlantAngiosperm, Callicarpa americana, Capsicum, Craterostigma, Eragrostis nindensis, Hyssopus officinalis, Lpomoea purpurea, Nepeta cataria, Nepeta mussinii, Oryza, Pennisetum glaucum, Petrea volubilis, Prunus canescens, Prunus cerasus, Prunus fructicosa, Rice, Salvia hispanica, Solanaceous, Yucca brevifolia (Joshua tree), Zea mays
Microbial Multiplexing
BacteriaAcinetobacter, Chromobacterium, Enterobacter asburia, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Neisseria meningitidis, Rahnella, Rhodococcus rhodnii
YeastSaccharomyces spp.
Enrichment
xGen Lockdown Hybridization with IDT ProbesMouse (Berkeley laboratory line)

*Many other species have been sequenced at our core. Please contact us for more information, if needed.

Applications

I. Whole Genome Sequencing (https://www.pacb.com/applications/whole-genome-sequencing/)

II. Targeted Sequencing (https://www.pacb.com/applications/targeted-sequencing/)

III. Complex Populations (https://www.pacb.com/applications/complex-populations/)

IV. RNA Sequencing (https://www.pacb.com/applications/rna-sequencing/)

V. Epigenetics (https://www.pacb.com/applications/epigenetics/)