PacBio Sequel 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.

Sequel System applications:

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 Noah Workman (jnworkman@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 SMART 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.) 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 (significantly higher than the desired insert size in the library) without any smear of degradation products.

For 20 Kb SMRT library, the DNA should be higher than 30 Kb, and for 10-15Kb SMRT library, the DNA should be larger than 20 kb. DNA purity is crucial for successful sequencing. The 260/280 ratio should be around 1.8 (+/-  0.2) and the 260/230 should be in the 2:2.2 range.

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

Buffer and concentration:  DNA or RNA should be in 10 mM Tris, pH 7.5-8.0 at a minimum concentration of 50-100ng/µl.

Required DNA and RNA quantities and concentration:

Sample concentration = 50-100 ng/ul as quantitated by QUBIT

If you are 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 AND NOT ROUND/FLAT BOTTOM PLATES.

Table 1. Sample Requirements

Library Size and Type Amount Required 
DNA10-20, 20-30 Kb
1-5 Kb

250-500 bp
DNA


Amplicons
4 µg
4 µg

500-750 ng
RNA (IsoSeq)1-2, 2-3, 3-6, 6-10 Kb cuts

No cuts
Total RNA2ug

2ug


 

 

PacBio Guidelines for Successful SMRTbell Libraries

Q&A for “DNA Quality Requirements for Single Molecule, Real-Time (SMRT) Sequencing”

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.

Table 1. PacBio Sequel DNA library preparation and SMRT cell run fees

ServiceUGA FeeNon-UGA FeeCommercial Fee
DNA quality assessment for PacBio library
$37.00$43.70$55.50
Large SMRTbell library (15-20kb and >30kb)
$533.00$629.00$800.00
Multiplexed SMRTbell genomic libraries pre-pooling (per sample)
$216.00$255.00$324.00
Multiplexed SMRTbell genomic libraries post-pooling (per pool)
$166.00$196.00$249.00
Quality assessment of final SMRTbell library/pool
$29.00$34.25$43.50
DNA library binding, annealing, and cleanup prior to sequencing
$211.00$249.00$317.00
PacBio Sequel SMRTCell sequencing run (10-hr movie)
$1,108.00
$1,308.00
$1,662.00

Table 2. PacBio Sequel 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
$267.00
$316.00
$401.00
PacBio Sequel SMRTCell sequencing run for IsoSeq (20-hr movie)
$1,415.00
$1,670.00
$2,123.00

Table 3. PacBio Sequel custom methods

ServiceUGA FeeNon-UGA FeeCommercial Fee
Library from amplicons (500 bp-4 Kb)Please inquirePlease inquirePlease inquire
PacBio: xGen Hybridization using IDT probes$748$883$1,122
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. List of the Species Sequenced on the PacBio Sequel

Order TypeSpecies/OrganismScientific name (if provided by customer)
IsoSeqAzaleaRhododendron canescens
Large Molecule Library, WGSMaizeZea mays
Microbial Multiplexing, WGSMeningitidis bacteriaNeisseria meningitid
Microbial Multiplexing, WGSE. coliE. coli
Microbial Multiplexing, WGSE. asburiaE. asburia
Microbial Multiplexing, WGSE. cloaceE. cloace
Microbial Multiplexing, WGSK. pneumoniaeK. pneumoniae
IsoSeqDrosophilaDrosophila recens
IsoSeqDrosophilaD. subquinaria
IsoSeqBipalium kewenseBipalium kewense
Large Molecule Library, WGSLobsterHomarus americanus
Microbial Multiplexing, WGSYeastSaccharomyces spp
Large Molecule Library, WGSMorning GloryIpomoea purpurea
Microbial Multiplexing, WGSBacteriaRhodococcus rhodnii
Microbial Multiplexing, WGSBacteriaChromobacterium sp.
Microbial Multiplexing, WGSBacteriaAcinetobacter sp.
Microbial Multiplexing, WGSBacteriaRahnella sp.
Xgen Lockdown Probe SequencingMouseBerkeley laboratory line
IsoSeqGerbilMongolian gerbil
Large Molecule Library, WGSFungusGanoderma
Large Molecule Library, WGSFungusPleurotus flabellatu
Large Molecule Library, WGSC. elegansC. elegans
Large Molecule Library, WGSgreen algaPicocystis salinarum
IsoSeqmosquitoAnopheles stehpensi
Large Molecule Library, WGSheartwormDirofilaria immitis
Large Molecule Library, WGSBacteriaBifidobacterium
Large Molecule Library, WGSRiceOryza sp
Large Molecule Library, WGSPetrea volubilisPetrea volubilis
Large Molecule Library, WGSCallicarpa americanaCallicarpa americana
IsoSeq synthetic RNA
Large Molecule Library, WGSSalvia hispanicaSalvia hispanica
Large Molecule Library, WGSHyssopus officinalisHyssopus officinalis
Large Molecule Library, WGSNepeta mussiniiNepeta mussinii
Large Molecule Library, WGSNepeta catariaNepeta cataria
Large Molecule Library, WGSJoshua TreeY. brevifolia
Large Molecule Library, WGSL. subsacemosaL. subsacemosa
Large Molecule Library, WGSL. brevisandL. brevisand
IsoSeq Pitcher PlantPurpurea_Rosea
IsoSeq Pitcher PlantPsittacina
Large Molecule Library, WGSFungusCandida albicans
Large Molecule Library, WGSFungal plant pathogenFusarium virguliforme
Large Molecule Library, WGSAngiosperm
IsoSeqCopepodsNeocalanus flemingeri
IsoSeqCopepodsPseudocalanus spp.
IsoSeqCopepodsMetridia pacifica
Large Molecule Library, WGSBacterial pathogen of honey beeSerratia sp.
Large Molecule Library, WGSgrassEragrostis nindensis