Pacific Biosciences Sequencing Submission

Updated 3-14-25

Long Read Sequencing Technology

Long-read sequencing has revolutionized genomics by enabling the generation of contiguous, high-fidelity sequences that span complex genomic regions, overcoming the limitations of short-read technologies. By producing reads that extend tens of kilobases, long-read platforms facilitate the assembly of complete genomes, the resolution of structural variations, and the accurate phasing of haplotypes. Among the leading technologies, Pacific Biosciences (PacBio) stands out for its Single Molecule, Real-Time (SMRT) sequencing, which offers high accuracy and ultra-long reads, making it a powerful tool for applications ranging from de novo genome assembly to epigenetic analysis.

PacBio Revio Technology

Pacific Biosciences's SMRT (Single Molecule Real Time) sequencing technology starts with the ligation of SMRTbell adapters to double stranded DNA forming a circularized template, followed by the addition of primers and polymerase to the DNA template. The SMRTbell libraries are then loaded to a SMRT cell, and sequenced on PacBio's Revio instrument. Each Revio SMRT cell contains 25 million nanowells, called ZMW's (zero-mode waveguides), where fluorescence events corresponding to the addition of a specific nucleotide by the polymerase tethered to the bottom of the ZMW are detected. Revio runs up to 8 SMRT cells in parallel with movie times lasting up to 30 hours.

Higher Accuracy with HiFI Reads

High accuracy reads can be achieved by Circular Consensus Sequencing (CCS) where 1.) a polymerase reads around the same circularized DNA multiple times; 2.) polymerase reads are trimmed off adapters to yield subreads; 3.) subreads are collapsed into a single consensus read; 4.) a HiFi read is generated from >=3 CCS reads with accuracy >=99%. For optimal sequencing, libraries should be no longer than ~20Kb in length.

How to Get Started with PacBio

If you are new to SMRT sequencing technology we recommend that you visit PacBio's website to explore the different applications and library preparation methods available based on the scientific questions that you are trying to answer. If you still have questions after reading over the website, you can reach out to a PacBio Scientist and/or reach out to the CZ Biohub SF Genomics Platform team at seq-team@sf.czbiohub.org.

Here are some resources:

• Overview-HiFi-Application-Options

Here are some questions to get you started:

• What PacBio application will help answer your scientific question (e.g. WGS, targeted sequencing, amplicons, RNA sequencing, metagenomics, viral sequencing)?
• What library preparation protocol is best for the type and quantity of starting material that you have?
• How many reads do you need? This will dictate how many 8M Revio SMRT cells to load. The expected yield per Revio SMRT cell run is ~ 8M reads.

Sequencing Submission Overview

We currently only accept prepared SMRTbell libraries. If you have questions about what library protocol to use based on your application of interest please refer to PacBio's sequencing application guide (see Procedure and Checklist Reference). If you are multiplexing samples and need a set of SMRTbell barcodes, please include this information in the sample sheet.

Please create an initial submission and fill out the sample sheet prior to dropping off the library pool at CZ Biohub SF in San Francisco.

Library pool requirements

25 μL at 30-60 ng/μL is enough for quality check and sequencing on 1 Revio SMRT cell. The expected yield per 1 SMRT cell is ~ 8M reads, so plan the number of SMRT cells that you need, and/or the number of libraries that you plan to multiplex accordingly. Please send your libraries in 1.5mL tubes, we do not accept any other format.

Dropping off samples

Samples should be dropped off at (or shipped to) the CZ Biohub SF location in Mission Bay. The address is:

    Chan Zuckerberg Biohub SF
    Attn: Genomics Platform
    499 Illinois St, 4th floor
    San Francisco, CA 94158

Once you enter the lobby of 499 Illinois St., check in with personnel at the front desk. Someone will lead you to the sample drop box located in the mail room on the ground floor. Scan the QR code on the box, enter your information (Name, library name, # libraries, etc.) to register the sample drop off and place the 1.5 ml tube through the top front insert on the metal box. Please do not place extra ice, or dry ice in the box.

Please note: drop-offs are on Mondays and Wednesdays 9AM-12PM only.

SMRTbell library QC

Once we receive your library we will perform our own quality check before loading the library on the Revio. We will reach out to the submitter via email if we need further information.

Sequencing turnaround time

The turnaround time for sequencing is dependent on the number of SMRT cell requests in our queue. We usually sequence 4 SMRT cells in one continuous run. The actual run may take from 2-5 days. Post analysis and data delivery may take another 1-2 days. Once the library is loaded, expect a turnaround time of 10 days for data delivery.

Data Delivery

An AWS token will be provided for data download. For first time collaborators, please make sure that you have AWS CLI installed on your computer. The token is valid for 36 hours, so please download your data as soon as you receive an email from the Genomics Platform team containing the token.

Sample submission form

Once you have read the instructions above, please click the button below to proceed to the sample submission form.

Proceed to the submission form