PacBio Sequel II Sequencing

Sequel 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.

Applications:

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

Consultation and Assistance

Contact for Genomics and Bioinformatics Consultation

Please use this link to send an email to Dr. Magdy Alabady for consultation on new or existing Genomics and Bioinformatics projects. Also, you can contact Dr. Alabady for assistance with grant proposals and for obtaining a letter of support from GGBC.

Contact for PacBio Technical Assistance

Please contact Contact Tyler Simmonds (tsim0604@uga.edu), Lab Manager, for technical questions about existing or new Sequel projects.

More contacts

Please visit our “All Inquiries” page for detailed information about who you should contact at GGBC to receive a quick and accurate response.

Sample Preparation

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 directly used as templates for the sequencing process. The quality of the DNA and RNA starting material will be directly reflected in 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.

Required quantitation method:

For DNA, please use pico green or equivalent fluorometric method (e.g., QUBIT).

For RNA, please use either ribo-green or QUBIT.

DNA quality: DNA should be high molecular weight without any smear of degradation products. For best results it is imperative that the input DNA size is significantly higher than the desired insert size of the final library. For 20 Kb SMRT libraries, the DNA should be higher than 30 Kb, and for 10-15Kb SMRT library, the DNA should be larger than 20 kb. Longer read lengths can be achieved with higher input amounts of sufficiently high molecular weight input.

DNA purity is especially crucial for successful sequencing with PacBio. The 260/280 ratio should be around 1.8 (+/-  0.2) and the 260/230 should be in the 2:2.2 range. Significant contaminants that cannot be removed by bead cleanup can cause library prep and/or sequencing to fail.

RNA quality: Good quality, intact RNA is essential. RIN must be ≥8.0 as indicated by the Agilent Bioanalyzer.

Buffer: DNA or RNA should be in 10 mM Tris, pH 7.5-8.0. Water is okay if it is nuclease free, but buffer is preferred.

Required DNA and RNA quantities and concentration:

Standard DNA library prep: a minimum of 4 ug of DNA per sample is required. Please submit samples in single lo-bind tubes in a volume of 100 uL or less. Higher volumes will require concentration into a smaller volume.

Standard DNA library prep with aggressive size selection for the longest possible read lengths (>30 kb): minimum10 ug of DNA per sample is required, higher amounts are recommended. Please submit samples in single lo-bind tubes in a volume of 100 uL or less. Higher volumes will require concentration into a smaller volume. If your DNA is sufficiently high molecular weight and you can provide enough input material, we recommend providing significant excess DNA for aggressive size selection to achieve longer read lengths.

Low input DNA library prep: a minimum of 150 ng of DNA per sample is required. Please note that size selection cannot be done with this low input protocol, and we recommend providing more material for standard library prep if possible. This option is only recommended for samples types for which it is difficult to acquire higher amounts of DNA, i.e. single insects.

RNA for IsoSeq: 10 uL of RNA at least 250ng/uL is required. Lower input amounts will require several replicates and increased amplification.

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.

Table 1. Sample Requirements

Library TypeLibrary Insert SizeAmount Required
DNA10 kb, 15kb, 20 kb, 30 kb4 µg
DNALow Input, no size selection4 µg
DNAAmplicons500-750 ng
DNAAggressive size selection, > 30kb10 µg or more
RNA 2µg

 

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

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.

Table 1. Full-length Barcoded Amplicons Custom/16S/18S/ITS

ServiceUGA FeeNon-UGA FeeCommercial Fee
Full-length Barcoded Amplicons of custom/16S/18S/ITS (1-48 samples)$600$708$900
Full-length Barcoded Amplicons of custom/16S/18S/ITS (49 -96 samples)$1,000$1,180$1,500
Library from Amplicons$533$629$800
Quality assessment of final SMRTbell library/pool$29$34.25$43.50
DNA library binding, annealing, and cleanup prior too sequencing$230$272$345

Table 2. PacBio Sequel II DNA library preparation and SMRT cell run fees

ServiceUGA FeeNon-UGA FeeCommercial Fee
DNA quality assessment for PacBio library
$37$43.70$55.50
Large SMRTbell library (15-20kb and >30kb)
$533$629$800
Multiplexed SMRTbell genomic libraries pre-pooling (per sample)
$216$255$324
Multiplexed SMRTbell genomic libraries post-pooling (per pool)
$166$196$249
High Fidelity Library (CCS; includes SageELF run)$713$842$1,070
Quality assessment of final SMRTbell library/pool
$29$34.25$43.50
DNA library binding, annealing, and cleanup prior to sequencing
$230$272$345
PacBio Sequel SMRTCell sequencing run for CLR (15 hrs)
$1,774$2,094$2,661
PacBio Sequel SMRTCell sequencing run for HiFi (32 hrs)
$2,400
$2,832
$3,600

Table 3. PacBio Sequel II IsoSeq library preparation and SMRT cell run fees

ServiceUGA FeeNon-UGA FeeCommercial Fee
RNA quality assessment for PacBio library
$86.00
$102.00
$129.00
IsoSeq Library
$627.00
$740.00
$941.00
Multiplexed IsoSeq libraries pre-pooling (per sample)
$424.00
$501.00
$636.00
Multiplexed IsoSeq libraries post-pooling (per pool)
$204.00
$241.00
$306.00
Quality assessment of final IsoSeq SMRTbell library/pool
$28.00
$33.05
$42.00
IsoSeq library binding, annealing, and cleanup prior to sequencing
$230.00
$272.00
$345.00
PacBio Sequel SMRTCell sequencing run for IsoSeq (26 hrs)
$2,100.00
$2,478.00
$3,150.00

Table 4. PacBio Sequel custom methods

ServiceUGA FeeNon-UGA FeeCommercial Fee
HMW DNA extraction for PacBio/10x Genomics$220$260$275
PacBio: xGen Hybridization using IDT probes$748$883$935
Data Retrieval
  1. Primary analysis files will be transferred to customers through the GGBC’s data distribution FEX server. The following files are an example of the primary analysis output files: • *.adapters.fasta • *.scraps.bam.pbi • *.subreads.bam • *.subreadset.xml • *.txt • *.scraps.bam • *.sts.xml • *.subreads.bam.pbi • *.transferdone.

 

  1. The primary analysis files will be permanently deleted from GGBC’s data storage after 6 months from the data transfer date.
Sequenced Species

Table 1. Organisms Sequenced on the PacBio Sequel at GGBC

IsoSeq 
AzaleaRhododendron canescens
CopepodsNeocalanus flemingeri, Pseudocalanus, Metridia pacifica
FishGasterosteus aculeatus
GerbilMongolian gerbil
MosquitoAnopheles stehpensi
NematodeBipalium kewense
PlantBipalium kewense
Synthetic RNA
Large Molecule Library
AlgaePicocystis salinarum
AnimalAnolis sagrei, Homarus americanus
BacteriaBifidobacterium, Serratia
FungiGanoderma, Hymenochaete, Oudemansiella, Phlebia, Pleurotus flabellatu, Trametes cubensis, Trametes elegans
InsectAphid, moth
NematodeCaenorhabditis elegans, Dirofilaria immitis
PlantAngiosperm, Callicarpa americana, Capsicum, Eragrostis nindensis, Hyssopus officinalis, Lpomoea purpurea, Nepeta cataria, Nepeta mussinii, Oryza, Petrea volubilis, Rice, Salvia hispanica, 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)