Home Products Pacbio RS Workflow Template Preparation

PACBIO RS WORKFLOW

Step 1: Sample Preparation

PacBio products required: DNA Template Prep Kit

The PacBio RS DNA Template Preparation Kit is used to convert your sample DNA into our proprietary SMRTbell™ library format for single molecule, real-time (SMRT™) sequencing. The SMRTbell DNA template preparation method creates a unique, structurally linear and topologically circular DNA morphology. This format allows for the sense and antisense compatibility required to achieve both standard and circular consensus sequencing on a single molecule. SMRTbell libraries are stable, and may be stored for up to six months after preparation.

 

Our DNA Template Preparation Kits provide a single streamlined protocol to create varied insert lengths, from 250bp to more than 6kb, for optimal performance with multiple sequencing protocols. Because a common polymerase initiation site is created using universal DNA-ligated hairpin adapters, insert size is not a constraint. This approach also reduces sequencing bias and normalizes performance across variable insert sizes.

 

 

The DNA Template Preparation protocol is compatible with a variety of sample sources and front-end pathways. Whether accomplished on the benchtop or with robotic liquid-handling, the process is simple and streamlined. There is no need for routine amplification and only 500ng of sample is required for libraries of 1kb or smaller. Suitable sample types include: purified genomic DNA, BACs, cDNA libraries and PCR products. In cases of insufficient starting material, or derivation from an alternative source such as tissue, whole genome amplification (WGA), target enrichment and cDNA isolation protocols can be used.

Step 2: Run Design

Products needed for this step: RS Remote

Following preparation of your SMRTbell™ template library, you are ready to customize your sequencing run using the RS Remote.

The RS Remote is a Windows®-based application that allows you to design and monitor PacBio RS sequencing runs directly from your desktop or other remote location. Use the RS Remote to design a new run, clone an existing run or import pre-defined experiment parameters from a comma-separated value (CSV) file. The RS Remote API function is also compatible with LIMS systems for more customized data management.

As you design a sequencing run, the RS Remote provides constant feedback regarding specific reagent requirements and expected time to completion. Once the run is complete, you can specify secondary analysis parameters through the RS Remote interface.

From run design and monitoring, to accuracy checks and analysis, the RS Remote guides you from beginning to end, from the convenience of your desktop.

Step 3: DNA Polymerase Binding

Products needed for this step: DNA/Polymerase Binding Kit

Once you have prepared your samples and customized your run design, you are ready to bind the SMRTbell™ DNA library to the polymerase in preparation for sequencing. This simple incubation step requires less than 30 minutes of hands-on time and the DNA/Polymerase complex is stable for up to 3 days.

Polymerase Binding

Step 4: Instrument Run

Products needed for this step: DNA Sequencing Kit 
                                                        SMRT Cells
                                                        PacBio RS instrument 
                                                        RS Touch

Once the SMRTbell™ library is bound to the DNA polymerase, you are ready to prepare the PacBio RS system for sequencing.

The RS Touch is the integrated touchscreen interface that guides you through reagent loading and instrument operation. Based on your selected run design, the RS Touch makes the task of loading required consumables simple and error free. The intuitive screen displays the precise location of each loaded component and barcodes confirm accurate placement. Once the system validates proper preparation, you simply press start and walk away.

The PacBio RS automates the entire sequencing process. Driven by on-board intelligence and state-of-the-art optics and robotics, the system schedules, performs and monitors every step from SMRT Cell priming and polymerase immobilization to signal processing from thousands of ZMWs in real time.

Step 5: Primary Analysis

Products needed for this step: PacBio RS System
                                                        Blade Center

As automated sequencing progresses, real-time signal processing, base-calling and quality assessment are performed by the Blade Center. Through an optimized set of algorithms, the Blade Center translates the light pulses captured by PacBio RS optics into basecalls and associated quality metrics. This data, along with metrics such as readlength distribution and polymerase speed are available for immediate review through the RS Remote and RS Touch interfaces. Trace and pulse data associated with each SMRT Cell is also made available. This primary data is simultaneously streamed to the secondary analysis system for further interpretation.

Step 6: Secondary Analysis

Products needed for this step: SMRT Portal
                                                        SMRT View

Due to the nature of single molecule, real-time (SMRT™) sequencing, it is imperative that the secondary analysis keeps pace with the speed of the instrument. Pacific Biosciences delivers the secondary analysis tools capable of processing real-time sequencing data when you need it.

SMRT Portal
SMRT Portal is an open-source, browser-based application that allows you to create, submit and monitor secondary analysis jobs as well as view and download the results. With this tool, you can align reads to a reference sequence or assemble reads into a de novo genome. SMRT Portal also lets you creation and manage secondary analysis protocols and reference sequences manually. In addition to being able to manually set up secondary analysis through SMRT Portal, users have the option to automate secondary analysis directly through RS Remote.

SMRT Portal produces experiment specific, data-rich reports in industry-standard output formats containing both primary and secondary analysis data. Moreover, all data files are accessible directly through SMRT Portal, allowing for easy third party software analysis and colleague collaboration. A comprehensive web services APT exposes the full functionality of SMRT Portal for integration with your LIMS system and third party software.

     

SMRT View
SMRT View is a scalable and extensible application for graphical data exploration and discovery that allows you to visualize and interact with secondary analysis sequencing data. A flexible, application-oriented user interface provides you with a broad range of ways to navigate and explore the results of your experiments, supporting simple discovery of patterns in your data. Interactive graphical representations of variants, quality values, and other metrics, along with rich annotations allow you to rapidly validate your results by visualizing genome-scale data sets down to the SNP level. SMRT View, which includes out-of-the-box support for Sequencing and DeNovo workflows, is the first data visualization application that can visualize kinetics and structure information unique to PacBio's SMRT technology.



Step 7: Tertiary Analysis

Products needed for this step: PacBio DevNet
                                                        SMRT™ Compatible Directory

The PacBio RS system is designed as an open platform to support a wide array of APIs, File APIs and industry standard formats for your convenience. To facilitate tertiary analysis using common applications and support development of novel solutions, these interfaces and associated documentation are available at PacBio DevNet. Beyond the PacBio DevNet portal, we have developed the PacBio Compatible directory to catalog available third party software applications compatible for tertiary analysis of PacBio RS system data.

 A web of molecular interactions driving H2 production from Rhodopseudomonas Palustris.
A web of molecular interactions driving H2 production from Rhodopseudomonas Palustris