Pacific Biosciences Sequencing Submission

Long Read Sequencing

Long-read sequencing technology, also known as third-generation sequencing, allows for sequencing of native DNA molecules that are hundreds to hundreds of thousands of bases long. The advantages of doing long read sequencing over short-read (NGS) sequencing are numerous. For instance, sequencing of the native DNA molecule eliminates GC bias, amplification artifacts, and permits the direct detection of epigenetic markers. Long continuous reads lead to more accurate mapping of highly repetitive and complex regions. It also allows for higher sensitivity in variant detection, improved de novo assembly, transcript isoform identification and phased assembly -- the latter being the assignment of sequences to the appropriate chromosome set. Some disadvantages of this technology are the low throughput, higher error rate observed, and the need for a lot of high molecular weight DNA input, which current NGS extraction methods cannot provide.

PacBio Sequel IIe Technology

VIDEO

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 latest Sequel IIe instrument. Each SMRT cell contains 8 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.

Image Source: pacbio.com 

There are two modes of sequencing with PacBio:

  • Circular Consensus Sequencing (CCS) - the polymerase reads around the same molecule over and over again. CCS is suitable for inserts that are < 20 Kb long.

  • Continuous Long Read (CLR) - the polymerase reads around the long molecule one time around. This mode is used for DNA inserts that are > 20 Kb long.

Image Source: pacbio.com

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 question(s) 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 CZ Biohub’s Genomics Platform team.  

Here are some resources:

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)?
  • What library preparation protocol is best for the type and quantity of starting material that you have?
  • What sequencing mode is needed (e.g. CCS or CLR) based on your DNA insert size?
  • How many reads do you need? This will dictate how many SMRT cells to load. The expected yield per SMRT cell run is ~4M reads (~20-30 Gb).
  • What kind of secondary analysis will you need (e.g. demultiplexing, CSS, Iso-Seq Analysis)?

Sequencing Submission Overview

This submission system is for a Sequel IIe sequencer located at our San Francisco location.  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 dual index barcodes, please send an email to seq-team@czbiohub.org. 

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

  • Library pool requirements

The SMRTbell library concentration and volume needed will depend on the sequencing application, but in general 10-12 ul at ~50 ng/ul is enough for quality check and sequencing on 1 SMRT cell. The expected yield per 1 SMRT cell is ~4M reads (20-30 Gb), so plan the number of runs that you need, and/or the number of libraries that you plan to multiplex accordingly.

  • Ship or drop off* libraries (SF Mission Bay address):
    Chan Zuckerberg Biohub
    Attn: Genomics Platform
    499 Illinois St, 4th floor
    San Francisco, CA 94158

*Drop offs are Mondays and Wednesdays 9AM-NOON only.

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. An ice pack located inside the drop box will keep the samples cool.

  • SMRTbell library QC

Once we receive your library we will perform our own quality check before loading the library on the Sequel IIe. We’ll 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 SMRTbell libraries 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 day. Once the library is loaded, expect 1-1 ½  week turnaround time 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. 

  • Submission System Link

Please complete the sequencing submission below. Tooltips will provide more information if you hover over questions/fields. If you have any questions related to the submission system, please send an email to seq-team@czbiohub.org.