p a c b .c o m / w g s

PLANT AND ANIMAL SCIENCES

HUMAN BIOMEDICAL RESEARCH

Genomic concordance (QV) across the genic regions of multiple data sets of the rice genome assembled to a high contiguity. These data were generated on the Sequel II System with 1.0 chemistry and SMRT Link software v7.0.

FROM DNA TO REFERENCE-QUALITY GENOME

WHOLE GENOME SEQUENCING FOR DE NOVO ASSEMBLY BEST PRACTICES

WHOLE GENOME SEQUENCING

STRUCTURAL VARIANT DETECTION

Single Molecule, Real-Time (SMRT®) Sequencing on the Sequel® II System enables easy and affordable generation of high-quality de novo assemblies of even the most complex genomes. With megabase-size contig N50s, consensus accuracies >99.99%, and tools for phasing haplotypes you can capture undetected SNVs, fully intact genes, and regulatory regions embedded in complex structures that fragmented draft genomes often miss.

WORKFLOW RECOMMENDATIONS

- Start with unamplified genomic DNA >30 kb1

- 5 μg input for sequencing ≥2 SMRT Cells 8M

- Prepare long-insert libraries using the SMRTbell® Express Template Prep Kit 2.02

- Low DNA input workflow available for small genomes on the Sequel System3

- Size select library for optimal yield and read lengths4

- Sequence to ≥30-fold unique molecular coverage5 per haplotype in the continuous long read (CLR) sequencing mode*

- Average yields of 75 Gb and up to 160 Gb per SMRT Cell 8M

- Utilize the PacBio analytical portfolio for genome assembly6

CONSENSUS ACCURACY >99.99% (QV40)

Shear DNA

Adapter Ligation

DNA Repair

Size Selection

Prepare Template for Sequencing

Sequence on Sequel II System

Analyze with SMRT Analysis or

PacBio DevNet Tools

20

25

30

35

40

45

50

5 10 15 20 25 30 40 50 60

Con

cord

ance

QV

Total Fold Coverage

*Highly accurate long reads (HiFi reads) generated using the circular consensus sequencing (CCS) mode on the Sequel II System can also be used for whole genome sequencing and assembly. This approach has currently been tested on a limited number of samples; for more information on how to use this methodology, please contact your local Field Application Scientist.

KEY REFERENCES1. Technical Note: Preparing DNA for PacBio Whole Genome Sequencing for De Novo Assembly: Quality Control and Shearing

2. Procedure and Checklist – Preparing gDNA Libraries Using the SMRTbell Express Template Preparation Kit 2.0

3. Application Note: Low DNA Input Workflow Considerations for De Novo Assembly

4. Technical Note: Preparing Libraries for PacBio Whole Genome Sequencing for De Novo Assembly: Quality Control and Size Selection

5. PacBio Assembly Tool Suite: FAQ – What Coverage do I Need for De Novo Assembly and Polishing?

6. PacBio Analytical Portfolio for Whole Genome Sequencing

7. De Novo Assembly Brochure: Assembly Options for Your SMRT Sequencing Data

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2019, Pacific Biosciences of California, Inc. All rights reserved. Information in this document is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this document. Certain notices, terms, conditions and/or use restrictions may pertain to your use of Pacific Biosciences products and/or third party products. Please refer to the applicable Pacific Biosciences Terms and Conditions of Sale and to the applicable license terms at http://www.pacb.com/legal-and-trademarks/terms-and-conditions-of-sale/. 

Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science. NGS-go and NGSengine are trademarks of GenDx. Femto Pulse and Fragment Analyzer are trademarks of Agilent Technologies Inc. All other trademarks are the sole property of their respective owners. PN: BP102-041219

The PacBio analytical portfolio offers solutions for every user in the lab from easy-to-use interfaces to command line tools and full-service genomics.

Assembly graphs from FALCON-Unzip (top) and FALCON-Phase (bottom), where different colors represent phase blocks, demonstrating near complete phasing of genomes.

Plant and vertebrate genome assemblies generated with SMRT Sequencing data from the genome database on NCBI showing PacBio assemblies readily achieve contig N50s ≥1 Mb.

DATA ANALYSIS SOLUTIONS WITH THE PACBIO ANALYTICAL PORTFOLIO

- Generate highly accurate de novo assemblies with megabase-level contiguity7

- Push-button assembly for small genomes with HGAP4 using SMRT Analysis

- Phased assembly at the command line with FALCON, FALCON-Unzip, and FALCON-Phase from PacBio DevNet

- Network of analysis partners for platform or full-service bioinformatics

HAPLOTYPE PHASING FOR DIPLOID ASSEMBLY HIGH CONTIGUITY ACROSS GENOME SIZES

FALCON; FALCON-Unzip;FALCON Phase

HGAP4

Analysis Partners