AAAAAAAAAAAAAAADDDDDDDDDDDDDDDDDDDDDDDDDDDDDD AAAAAAAAAAAAAA NNNNNNNNNNNNNNEEEEEEEEEEEEEEWWWWWWWWWWWWWWW DDDDDDDDDDDDDDDIIIIIIIIIIIIIIMMMMMMMMMMMMMMMEEEEEEEEEEEEEENNNNNNNNNNNNNNSSSSSSSSSSSSSSSIIIIIIIIIIIIIIOOOOOOOOOOOOOONNNNNNNNNNNNNNN TTTTTTTTTTTTTTOOOOOOOOOOOOOO YYYYYYYYYYYYYOOOOOOOOOOOOOOUUUUUUUUUUUURRRRRRRRRRRR

RRRRRRRRRRRRRRRRRREEEEEEEEEEEEEEEEEESSSSSSSSSSSSSSSSSSSEEEEEEEEEEEEEEEEEEEAAAAAAAAAAAAAAAAAARRRRRRRRRRRRRRRRRRCCCCCCCCCCCCCCCCCCHHHHHHHHHHHHHHHHH WWWWWWWWWWWWWWWWWIIIIIIIIIIIIIIIIITTTTTTTTTTTTTTTTTHHHHHHHHHHHHHHHHHH SSSSSSSSSSSSSSSSSMMMMMMMMMMMMMMMMMMRRRRRRRRRRRRRRRRRRTTTTTTTTTTTTTTTTTT®®®®®® SSSSSSSSSSSSSSSSSSSEEEEEEEEEEEEEEEEEEEEEQQQQQQQQQQQQQQQQQQQQQUUUUUUUUUUUUUUUUUUEEEEEEEEEEEEEEEEENNNNNNNNNNNNNNNNNNCCCCCCCCCCCCCCCCIIIIIIIIIIIIIIINNNNNNNNNNNNNNNNNNGGGGGGGGGGGGGGGGG

EpigeneettiiccsGenome finishing Haplotyyppee phasing• ••

Repeat expansions IIssoofform sequenciinngg Minor variants•• •

The PacBio RS II finishes microbial genomes and improves assembly of larger organisms with multi-kilobase reads and unbiased coverage regardless of GC content. No amplification is required.

Extraordinary Read Lengths with the PacBio RS IIThe PacBio® RS II sequencing system allows scientists to rapidly and cost effectively generate finished genome assemblies, reveal and understand epigenomes, and characterize genomic variation. It achieves the industry’s longest read lengths and highest consensus accuracy.

Generate Finished Genomes

www.pacb.com/denovo

Paper: Genome Biology: Reducing assembly complexity of microbial genomes with single-molecule sequencing

Paper: Nature Methods: Non-hybrid, finished microbial genome assemblies from long-read SMRT sequencing data

Paper: PloS One: Mind the gap: Upgrading genomes with Pacific Biosciences RS long-read sequencing technology

Paper: Applied and Environmental Microbiology: Genome of the anaerobic fungus Orpinomyces sp. C1A reveals the unique evolutionary

history of a remarkable plant biomass degrader

Range of Genome Sizes

Lower Eukaryotes Higher EukaryotesViruses / BACs Bacteria

Benefits

• Resolve mobile elements and structural-variation events

• Generate complete, accurate, and contiguous genome assemblies

• Annotate more genes

De novo, finished assembly of Salmonella enterica subsp. enterica serovar Heidelberg, with predicted accuracy of > 99.999% (QV50) Tandem duplication

A B C

A B B C

1

2

2

1

Inversion

A B C

A B’ C

1

2

2

1

De Novo Assembly Methods

• Hierarchical Assembly

• Hybrid Assembly

• Scaffolding

• Gap Filling

PacBio long reads

Pre-assembled reads

Hierarchical Genome Assembly Process (HGAP)

Finished genome

Salmonella enterica serovar Heidelberg

4,783,952 bp

Plasmid 1110,366 bp

Plasmid 237,768 bp

Genome-wide detection of methylation for the German E. coli outbreak strain.

www.pacb.com/basemod

Methyltransferases bind specifically to DNA motifs in a genome and methylate bases. PacBio software locates

modified sites and motifs.

The PacBio® RS II detects DNA base modifications using the kinetics of the polymerization reaction during sequencing.

Discover the Epigenome

Methylome of the German E. coli outbreak strain. The inner and outer red circles show the kinetic signals. The colored internal tracks show the different methylation motif distributions.

70.5 71.0 71.5 72.0 72.5 73.0 73.5 74.0 74.50

100

200

300

400

Fluo

resc

ence

inte

nsity

(a.u

.)

Time (s)

CmA

AG TCT A A

6m

6m

Pol

ymer

ase

Dyn

amic

s

Forward Strand

Reverse Strand

4m4m

DNA

DNA Polymerase

Methyltransferase

SMRT® DNA Sequencing

6m

6m

6mA

DNA Methylation

4m

h l f

Analysis of Polymerase Kinetics

Motif Occurrence in Genome

Modified in Genome % Modified

5'-GATC-3‘ 3'-CTAG-5' 42,992 41,969 97.6%

5'-ACCACC-3‘ 3'-TGGTGG-5'

4,569 4,492 98.3%

5'-CTGCAG-3‘ 3'-GACGTC-5' 2,746 2,678 97.5%

5'-CCACN8TGAY-3‘ 3'-GGTGN8ACTR-5'

492 492

478 484

97.2% 98.4%

Paper: Current Opinion in Microbiology: Entering the era of bacterial epigenomics with SMRT DNA sequencing

Paper: Nature Biotechnology: Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using

single-molecule real-time sequencing

Paper: Nucleic Acids Research: The methylomes of six bacteria

Zero-ModeWaveguide

6mA

Characterize Genomic Variation

www.pacb.com/target

The PacBio® RS II’s long single-molecule reads and unbiased coverage provide access to the entire genome to accurately characterize genetic complexity.

Structural Variation and Copy-Number VariantsWith multi-kilobase reads, anchor the ends of duplicated and inverted regions of the genome, allowing direct analysis of structural variation.

Paper: American Journal of Respiratory Cell and Molecular Biology: Genome Reference and Sequence Variation in the Large Repetitive Central Exon of Human MUC5AC

Normal< 55 repeats

Premutation55-200 repeats

Full mutation> 200 repeats

CGG repeat

FMR1 gene

Repeat ExpansionsSpan extreme CGG repeats and AT-rich regions with minimal bias, over hundreds to thousands of bases.

Paper: Genome Research: Sequencing the unsequenceable: Expanded CGG-repeat alleles of the fragile X gene

Isoform SequencingFull-length sequencing of intact transcripts eliminates the need for read assembly, identifies splice variants, and improves gene annotation, to better understand the expressed gene set.

Poster: ABRF 2013: Full-length cDNA sequencing on the PacBio RS

A A A T T T G ATTA A T T G C A A A TT G C T G G T C C C T A T G T C C T G T ATT TTTAAAA T GGG TTT GG ATTA AA AAAA A A TTTT TTTTTTTT TTTT CCCCCCCCC CC T G T AHG19

PacBio

Sanger

SNP Detection and ValidationHighly accurate SNP validation for any genomic region reduces false positives and false negatives.

Paper: BMC Genomics: Pacific Biosciences sequencing technology for genotyping and variation discovery in human data

SNP254 C>A

% Mismatch expected

% M

ism

atch

cal

cula

ted

0.0

0.0

0.5

1.0

1.5

0.5 1.0 1.5

Minor Variants and QuasispeciesSingle molecule sequencing simplifies the analysis of mixed populations of sequences. Exquisitely sensitive and specific.

Poster: CROI 2013: Sensitive detection of minor variants and viral haplotypes using SMRT sequencing

Full-Length 16S SequencingImprove taxonomic resolution on metagenomic samples by sequencing full-length rRNA genes (16S, 18S) or other common species markers such as rpoB.

Poster: ASM 2013: Analysis of Full-Length Metagenomic 16S Genes by SMRT® Sequencing

3.0%

Minor Variant Percentage

6.2%

29.6%

Internal Tandem Duplication

Val

Leu

Tyr

1,400 bp

FLT3 gene model in acute myeloid leukemia

Compound Mutations and Haplotype Phasing

Study linked mutations hundreds, even thousands, of bases apart.

Paper: Nature: Validation of FLT3-ITD as a therapeutic target in human acute myeloid leukemia

Linear variant detection to < 0.1% frequency

Cecum

15 229 137 318 100 684Water Soil

PacBio® RS II Typical Results

P6-C4 Chemistry

0

500

1000

1500

2000

2500

3000

3500

4000

1000 6000 11000 16000 21000 26000 31000 36000

Rea

ds

Read Length

Based on data from a 20 kb size-selected E. coli library using a 4-hour movie.Each SMRT Cell yields ~ 50,000 reads.

Half of data in reads: > 14 kb

Top 5% of reads: > 24 kb

Maximum read length: > 40 kb

Data per SMRT® Cell: 500 Mb – 1 Gb

Template Preparation Accuracy

Insert Size (bp) Typical Input DNA per Prep (ng)*

250 – 500 250

1,000 – 2,000 500

5,000 – 10,000 1,000

15,000 – 20,000 (size-selected) 5,000

*Low input options (10 – 100 ng) available on SampleNet.Each library prep typically supports > 35 SMRT Cells.

5 μg input material yields a minimum of > 16 SMRT Cells with a size-selected library.

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 20 40 60 80 100

QV

Coverage Aligned to Reference

Perfect consensus

Data generated with P6-C4 chemistry on PacBio® RS II;Resequencing analysis using SMRT Analysis v2.3.

The PacBio RS II sequencing provides average read lengths in excess of >10 kb with ultra-long reads > 40 kb. The long reads are characterized by high 99.999% consensus accuracy, and are ideal for de novo assembly, targeted sequencing applications, scaffolding and spanning structural rearrangements.

Products

Reagents Instrument Run Data Analysis

SMRTbell™ Template Prep Kit

DNA Polymerase Binding Kit

MagBead Kit

AMPure® PB beads

PacBio RS II with touch screen

RS Remote for run design

SMRT Cells

DNA Sequencing Kit

SMRT Analysis

SMRT Portal

SMRT View

Sequencing time30 to 240 min per SMRT Cell

From DNA to data in as few as 10 hours

No amplification required

Automation friendly

Open source Open standards

The PacBio® RS II system, consumables and software provide a complete set of tools to quickly and easily perform cutting-edge molecular biology.

SMRT® TechnologyThe PacBio RS II is based on novel Single-Molecule, Real-Time (SMRT) technology, which enables the observation of DNA synthesis by a DNA polymerase in real time. Sequencing occurs on SMRT Cells, each containing thousands of Zero-Mode Waveguides (ZMWs) in which polymerases are immobilized. The ZMWs provide a window for watching the DNA polymerase as it performs sequencing by synthesis.

SMRT Cells Phospholinked Nucleotides

Zero-Mode Waveguides

Primer

TemplatePolymerase

For Research Use Onlnly.y Not for use in diiagagnosticc pprocedures. © Copyright 2014, Pacific Biosciences of California, Inc. All rightsreserved. Information inn tthis document is suubjbject to cchah nge without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this docummenent. Certain notices, teterms, conondid tions and/or use restrictions may pertain to your use of Pacific Biosciences

j g p y y

products and/or third party prododucu ts. Please refer ttoo ththe appllicicaba le Pacific Biosciences Terms and Conditions of Sale and to the y p y

applicable license terms at http:///w/wwww .pacificbiosciencess.coc m/liceensnses.html.p p y p ppp

Pacific Biosciences, the Pacific Biosciencnces logo, PacBio, SMRRTT, SSMRTbelelll anand Iso-Seq are trademarks of Pacific Biosciences in the United States and/or certain other countriess. AlA l other trademarks arere tthe sole prpropoperty of their respective owners.

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BR105-101514

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Operating EnvironmentInstrument and environmental cabinet

Power requirements: 208 – 240 VAC. UPS recommended Operating temperature: 15 °C – 25 °C (59 °F – 77 °F) ± 2 °C per hour Humidity: 20% – 80%, noncondensing Ventilation: HVAC capacity of up to 22,720 BTU (6654 Watts) Nitrogen: 90 – 125 PSI (4,654 – 6,464 torr) WxDxH: 78.9 in x 30.3 in x 62.2 in (200.4 cm x 77.0 cm x 158.0 cm) Weight: 2,405 lb (1,091 kg)

Blade Center

Includes integrated computation and storage for performing single molecule, real-time sequencing, basecalling and quality assessment.

WxDxH: 24.1 in x 35.9 in x 26.2 in (61.3 cm x 91.3 cm x 66.5 cm) Weight: 300 lb (136 kg)

PacBio Community

pacbiodevnet.com

Open-source software, data, documentation and tips for PacBio sequencing

pacbiosamplenet.com

PacBio DevNetOpen repository for community-developed sample prep methods, PacBio early-release protocols, and official PacBio protocols

PacBio SampleNet