PacBio WGS

PacBio WGS PacBio AAV PacBio IsoSeq

The PacBio WGS pipeline is the best option for large-scale, complex eukaryotic genomes such as amphibians, birds, reptiles, mammals, etc., and is also a great option for all organisms when aiming for reference-quality assemblies. We generally recommend a minimum target depth of 15-20x for data that can later be assembled into publication-quality genomes. 

Our PacBio WGS pipeline uses PacBio’s SMRTbell prep kit 3.0 for circularized library prep and relies on the Megaruptor 3 shearing technology with adjustable fragment length to produce HiFi reads via CCS. This library preparation and sequencing strategy is very sensitive to both DNA fragment size and sample contaminants. To produce high-quality samples compatible with this platform, we recommend careful consideration of extraction methods, fragment analysis before submission, and shipping on dry ice.  For more prep tips and expected fragmentation sizes, see the additional information below the services table. 

Uniform fragment size is a necessary part of producing HiFi reads for CCS. In order to balance this requirement with the goal of maximizing read lengths for each project, SeqCenter requires each project to be run on its own PacBio flowcell with multiplexing only libraries of the same sample type from the same customer. Each order will need to include one library preparation per sample and at least one flowcell, and every library preparation service is indexed to allow for multiplexing on any flowcell. 

Sample Requirements:

  • At least 1 µg total dsDNA
  • Maximum volume of 100 µL

What You Will Receive:

  • One fastq file for each sample
  • One bam file with run metric data
  • Sequencing stats summary
  • Materials and methods summary
  • Data shared via Box folder within 2 weeks

Packages & Pricing

Sequencing Package Price*
Sequel Ile 8M Flowcell
Run across a Full Flowcell. Library preparation separate.		 $1,800.00
Revio 25M Flowcell
Run across a Full Flowcell. Library preparation separate.		 $2,000.00
PacBio WGS Library Preparation
Includes QC and barcoding. One needed per sample. Sequencing separate.		 $250.00
*Orders >48 Samples receive 5% discount

To help our customers meet the strict sample requirements, SeqCenter offers convenient HMW DNA extractions using PacBio’s Nanobind PanDNA kit, which is specifically meant for use with PacBio library preparation. While we have had success with this kit with many bacterial samples, this kit is not broadly applicable. However, other options are available for some sample types where special considerations need to be made. See the extraction section below for more details.

We are able to accommodate all organisms up to BSL2+ and do not accept BSL3 or higher classified organisms for extraction services. Samples requiring DNA extraction can be submitted as aspirated cell pellets shipped frozen on dry ice or as growth on sealed nutrient agar plates shipped at ambient temperature. A minimum of 109 cells is recommended for all microbial DNA isolation submissions. We cannot culture samples. We encourage you to ensure samples are pure and that there is adequate growth on your plates before shipping.

PacBio naturally lends itself to genomic assembly projects, especially for complex and repeat-rich regions. We routinely produce well-annotated genome assemblies for bacterial and fungal samples and are happy to discuss the options available for collapsed assemblies of higher-order eukaryotes.

Assemblies also offer the opportunity for 4mC and 6mA methylation site predictions, though this goal must be noted before sequencing for the necessary data to be captured.

For additional information on our bioinformatic capabilities with PacBio data, please see our Bioinformatics Services page.

Considerations for Successful WGS PacBio Sequencing

The PacBio platform has some of the most stringent sample requirements of all of the DNA sequencing platforms we host. The extraction method and clean-up often have the greatest influence on sample quality in terms of purity and fragment length.

Beyond intolerance for the standard contaminants that can interfere with enzymatic reactions (like phenolates), common problem constituents that are carried over during extraction include RNA and polysaccharides. For samples with high RNA content, a simple RNase treatment is recommended before submission.

Polysaccharide contamination is a more difficult problem to remedy. Carryover is common for fungi, plant, insect, and crustacean samples, though it is possible for any organism with polysaccharides embedded in its cells, tissues, or extracellular matrices. Clean-up post extraction is more difficult than during, so we recommend choosing an extraction method that addresses this, such as a CTAB method or PacBio Nanobind extraction kit with necessary protocol modifications. PacBio’s technical support can provide modified protocols specific to your sample type.

Long starting fragment lengths optimize total output and fully utilize all of the benefits of the PacBio platform and prep. To minimize initial fragmentation, we recommend choosing an HMW (high molecular weight) extraction option for your sample, as these are often the gentlest methods. We strongly encourage this approach over more brute-force methods like bead beating. For particularly difficult samples, HMW extractions may require special modifications and extra considerations. We also strongly encourage customers to analyze fragment length before sample submission to assess how additional unavoidable fragmentation will affect read length.

Expected Fragmentation by Sample Type

When ordering PacBio WGS, we strongly encourage you to include your sample type (e.g., metagenomic, cell culture, origin, etc.), organism, and extraction method. This information will help us fine-tune the fragmentation process to provide maximum read length and total output. Below are some examples of sample types, typical extraction methods we see, and average read length that might be expected.

Sample Type Typical Extraction Method Expected Read Length
Metagenomic Bead-beating, physical lysis 2-6 kbp
Bacterial cell culture Enzymatic, column-based, or HMW 6-10 kbp
Plant HMW kit, Nanobind Plant kit Up to 18kbp

PacBio and Amplicon Libraries

The PacBio SMRTbell prep 3.0 kit can support sequencing of amplicon libraries with modifications to our standard workflow to remove fragmentation. Due to skew for overrepresentation of smaller fragments and possible flowcell failure, it is strongly recommended that library lengths differ by no more than 10%.

To ensure proper processing of amplicon libraries, please note that the sample is an amplicon library and that fragmentation should not be performed. For additional information, please contact us.

PacBio Sequencing and Modified Bases

PacBio’s SMRT technology is capable of capturing raw methylation kinetics that can later be used to predict 4mC or 6mA base modifications. In order to store this data, the sample must be flagged for this option in advance of sequencing, so customers must indicate that the optional data should be stored for their samples when placing an order. Post-sequencing, predictions can be made in relation to an assembly but cannot be placed on individual reads. For additional information about SeqCenter’s add-on services for methylation calling, please see the PacBio Long Read portion of our Assembly and Annotation page.

Additional Resources

PacBio AAV

As a proud PacBio-sponsored AAV sequencing facility, we are excited to offer library preparation and sequencing for both ssAAV and scAAV samples. The platform’s CCS-generated HiFi reads are a great way to validate vectors and track new mutations.

Explore

PacBio IsoSeq

Novel isoform discoveries are within reach with high-quality, full-length mRNA sequencing. PacBio’s unique capabilities are prime for this growing field of study, allowing for detection of novel splicing arrangements possible with no assembly or inference needed. The HiFi reads also guarantee SNP detection for each form.

Explore

Contact:
91 43rd Street, Ste. 250
Pittsburgh, PA 15201
(878) 227-4915

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Full Flowcell:

We offer full flowcell sequencing services for libraries that have been prepared outside of our lab. For each library, we will need to know the loading concentration, phiX spike-in, and run structure. Data is delivered as a complete run folder and can be demultiplexed upon request if a sample sheet is provided.

SARS-CoV-2 Sequencing:

Our SARS-CoV-2 Sequencing services include an assembly on the SARS-CoV-2 reference genome as a scaffold. These packages provide roughly 200Mbp of paired end reads (2×151 bp) as fastq files and the assembly in fasta format.

We require a minimum sample volume of 15 µL of extracted material (or 250µL for VTM samples) and recommend that only samples with Ct values below 35 are sent (because submissions are not measured for viral load before processing). We offer services for both purified RNA and samples in VTM media that require viral RNA extraction. Please see our SARS-CoV-2 Sequencing page for more detailed information.

Microbiome Sequencing:

Please see our Microbiome Sequencing page for package details, including distributables and sample requirements.

Nanopore Sequencing:

Our Oxford Nanopore sequencing packages provide reads equal to the length of input DNA, delivered as a fastq file.

For each sample, we require a minimum volume of 60 µL, and at least 40 ng/µL dsDNA quantified by Qubit or other dye-based method (100 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Long Read Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

PacBio:

Our PacBio services deliver HiFi reads with an average read length of 8-12kb for standard offerings and up to 25kb for full flowcell runs. All package names are listed by the minimum number of >Q20 reads that will be delivered. Both assembly and annotation are included with all packages.

For each sample, a minimum volume of 60 µL and at least 35 ng/µL dsDNA (quantified by Qubit or other dye-based methods) are required. SeqCenter strongly recommends utilizing our HMW DNA Extraction service for best results; however, we will also accept samples that are shipped on dry ice to avoid DNA integrity issues caused by the shipping process.

Please see our PacBio Sequencing page for more detailed information about our services.

RNA Sequencing:

We offer both rRNA depletion RNA squencing and polyA-tail enrichment mRNA sequencing. Both provide stranded, paired end reads (2x 151bp) as fastq files.

For each sample, we require a minimum volume of 20µL and at least 50ng/µL RNA. We also recommend, but do not require, that samples be DNAse treated and have a RIN score greater than 6. We also offer Basic and Intermediate RNAseq analyses, which require an annotated reference sequence.

Please see our RNA Sequencing page for more detailed information about both our RNA sequencing and analysis packages.

Illumina Sequencing:

The Illumina sequencing packages produce paired end reads (2×151 bp) delivered as fastq files.

For each sample, we require a minimum volume of 30 µL and at least 10 ng/µL dsDNA quantified by Qubit or other dye-based method (20 ng/µL by Nanodrop). Variant calling add-on analysis requires an annotated reference file. DNA extraction services are available for only specific categories of microbes.

Please see our DNA Sequencing: Whole Genome Sequencing page for more detailed information about the package, variant calling analysis, and extraction requirements.

For additional information about variant calling and short read assemblies, please see our Bioinformatics page.

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