Quick Reference Card - Equipment and Materials …...De Novo – HiFi Low DNA De Novo Seq – Input Sequencing De Novo Length – hotgun Meta M icrobial Variant Detection Structural

Page 1 Part Number 100-815-000 version 03 (June 2020)

Equipment and Materials Needed for Sequencing Quick Reference Card

This Quick Reference Card lists examples of third-party laboratory equipment and other materials that are recommended for running specific types of applications on PacBio Sequel System and Sequel II System instruments. Note that although vendor part numbers are listed herein, they may not always be up to date. Verify and obtain the latest part numbers and relevant documentation from the specific vendor or manufacturer’s website. Laboratory Equipment and Material Categories

A. DNA Sizing Analysis B. DNA Quantification Analysis C. DNA Purity Analysis D. DNA Shearing E. DNA Size Selection F. General Laboratory Equipment G. General Laboratory Reagents and Consumables

SMRT Sequencing Application Categories Whole Genome Sequencing (WGS)

• De Novo Assembly – HiFi Reads • De Novo Assembly – Low DNA Input • De Novo Assembly – Microbial Multiplexing • Variant Detection • Structural Variant Detection

RNA Sequencing (RNA) • Iso-Seq Method • Single-Cell Iso-Seq

Metagenomics Sequencing (META) • Full-length 16S rRNA Sequencing • Shotgun Metagenomic Profiling or Assembly

Targeted Sequencing (TARG) • Amplicon Sequencing • No-Amp Targeted Sequencing

Laboratory Equipment and Material Recommendations Guide

+ Compatible with a specific application

‒ Not recommended for a specific application

Highly Recommended Where multiple compatible equipment options may be available for a particular category, specific products that are highly recommended are also highlighted


A. DNA Sizing Analysis

WGS RNA META TARG

Vendor Product PN

De

Nov

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Det

ectio

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Iso-

Seq

Sequ

enci

ng

Sing

le-C

ell I

so-

Seq

Sequ

enci

ng

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Bioanalyzer 2100 System for DNA Sizing Analysis Up To ~7 – 15 kb

Agilent Technologies

Bioanalyzer 2100 platform

Suitable for analysis of SMRTbell

libraries up to ~15 kb

G2939BA ‒ ‒ ‒ ‒ ‒ + + + ‒ + ‒


DNA 7500 Kit for Bioanalyzer 2100

5067-1506 ‒ ‒ ‒ ‒ ‒ ‒ ‒ + ‒ + ‒


DNA 12000 Kit for Bioanalyzer 2100

5067-1508 ‒ ‒ ‒ ‒ ‒ + + + ‒ + ‒


High Sensitivity Analysis Kit for Bioanalyzer 2100

5067-4626 ‒ ‒ ‒ ‒ ‒ + + + ‒ + ‒


A. DNA Sizing Analysis (Cont.)

WGS RNA META TARG

Vendor Product PN

De

Nov

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DN

A In

put

De

Nov

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Mic

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Varia

nt

Det

ectio

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Stru

ctur

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Varia

nt

Det

ectio

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Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Fragment Analyzer System for DNA Sizing Analysis Up To ~50 kb


Fragment Analyzer System


libraries up to ~50 kb

M5310AA ‒ ‒ ‒ ‒ ‒ + + + ‒ + ‒


Large Fragment 50 kb Kit for Fragment Analyzer

DNF-464-0500 ‒ ‒ ‒ ‒ ‒ + + + ‒ + ‒

Pippin Pulse System for DNA Sizing Analysis Up To ~100 kb

Sage Science Pippin Pulse System


libraries and genomic DNA up to ~100 kb

PPI0200 + ‒ + + + ‒ ‒ ‒ + ‒ +

Galileo Bioscience

Gel Tank with Tray and Combo 80-1214 + ‒ + + + ‒ ‒ ‒ + ‒ +

Sage Science 10X KBB Buffer KBB1001 + ‒ + + + ‒ ‒ ‒ + ‒ +

Lonza SeaKem® Gold Agarose 50150 + ‒ + + + ‒ ‒ ‒ + ‒ +


A. DNA Sizing Analysis (Cont.)

WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

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Low

DN

A In

put

De

Nov

o –

Mic

robi

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Varia

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tion

Stru

ctur

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nt

Det

ectio

n

Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Femto Pulse System for DNA Sizing Analysis Up To ~165 kb


Femto Pulse System

Rapid analysis time (<1.5 hours) Requires <1 ng of sample Suitable for analysis of SMRTbell

libraries and genomic DNA up to ~165 kb

M5330AA + + + + + + + + + + +


Genomic DNA 165 kb Kit for Femto Pulse

FP-1002-0275 + + + + + ‒ ‒ ‒ + ‒ +

CHEF Mapper XA System for DNA Sizing Analysis Up To ~10 Mb Bio-Rad Pulsed Field Gel Electrophoresis

System: CHEF Mapper® XA


libraries and genomic DNA up to ~10 Mb

170-3670 + ‒ + + + ‒ ‒ ‒ + ‒ +

Bio-Rad 10X TBE Electrophoresis Buffer

1610770 + ‒ + + + ‒ ‒ ‒ + ‒ +

Bio-Rad Pulsed Field Certified Agarose

1620137 + ‒ + + + ‒ ‒ ‒ + ‒ +

HIG

HLY

REC

OM

MEN

DED


B. DNA Quantification Analysis

WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

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Low

DN

A In

put

De

Nov

o –

Mic

robi

al

Varia

nt D

etec

tion

Stru

ctur

al V

aria

nt

Det

ectio

n

Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Fluorometric Assay Systems Thermo Fisher

Scientific

Qubit 4 Fluorometer

Fluorometric-based assay method

Enables highly accurate and sensitive quantitation of dsDNA

Q33238 + + + + + + + + + + +

Thermo Fisher

Scientific

Qubit Assay Tubes Q32856 + + + + + + + + + + +

Thermo Fisher

Scientific

Qubit 1X dsDNA High Sensitivity (HS) Assay Kit

Q33230 + + + + + + + + + + +

C. DNA Purity Analysis

WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

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DN

A In

put

De

Nov

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Mic

robi

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etec

tion

Stru

ctur

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aria

nt

Det

ectio

n

Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Spectrophotometric Assay Systems Thermo Fisher

Scientific

NanoDrop™ 2000/2000c Spectrophotometer

ND-2000 + + + + + + + + + + +


D. DNA Shearing

WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

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Low

DN

A In

put

De

Nov

o –

Mic

robi

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Varia

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etec

tion

Stru

ctur

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aria

nt

Det

ectio

n

Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

g-TUBE Devices for Shearing DNA to 6 – 20 kb Fragment Sizes Covaris g-TUBE

520079 + + + + + ‒ ‒ ‒ + ‒ ‒

Syringes & Needles for Shearing DNA to >30 kb Fragment Sizes Becton

Dickenson BD Luer-Lok™ 1-mL Syringe

309628 ‒ ‒ ‒ ‒ + ‒ ‒ ‒ ‒ ‒ ‒

SAI Infusion 26G Blunt End Needles

B26-150 ‒ ‒ ‒ ‒ + ‒ ‒ ‒ ‒ ‒ ‒

Megaruptor 2 System for Shearing DNA to 2 – 75 kb Fragment Sizes Diagenode Megaruptor 2 System

Long Hydropores for Megaruptor 2

Hydrotubes for Megaruptor 2

B06010002

E07010002

C30010018

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

‒ ‒ ‒

‒

‒ ‒

‒ ‒ ‒

+

+

+

‒ ‒ ‒

‒ ‒ ‒


D. DNA Shearing (Cont.)

WGS RNA META TARG

Vendor Product PN

De

Nov

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Low

DN

A In

put

De

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tion

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ctur

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aria

nt

Det

ectio

n

Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Megaruptor 3 System for Shearing DNA to 2 – 75 kb Fragment Sizes

Diagenode Megaruptor 3 System

Up to 8 samples can be processed in parallel without additional user input

Eliminates cross-contamination without requiring additional wash steps by using disposable shearing elements in a completely closed-system

Megaruptor 3 Shearing Kit

B06010003

E07010003

+

+

+

+

+

+

+

+

+

+

‒ ‒

‒

‒

‒ ‒

+

+

‒ ‒

‒ ‒

HIG

HLY

REC

OM

MEN

DED


E. DNA Size Selection

WGS RNA META TARG

Vendor Product PN

De

Nov

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DN

A In

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Det

ectio

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Iso-

Seq

Sing

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Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

SageELF System for DNA Size Selection 100 bp – 40 kb Sage Science SageELF System

Separates DNA samples by size,

and then simultaneously fractionates the whole sample into 12 fractions

One sample is fractionated on a single precast agarose cassette, and one or two cassettes may be processed at one time

ELF0001 + ‒ + + + ‒ ‒ ‒ ‒ ‒ ‒

Sage Science 0.75% Agarose Cassette for SageELF, 1 – 18 kb

ELD7510 + ‒ + + + ‒ ‒ ‒ ‒ ‒ ‒

BluePippin System for DNA Size Selection 100 bp – 50 kb Sage Science BluePippin System

Separates DNA samples by size,

and collects all fragments above a size threshold set by the user

Up to four samples (plus a Size Marker) can be fractionated on a single precast agarose cassette, and only one cassette may be processed at one time

BLU0001 + ‒ + + + ‒ ‒ ‒ ‒ ‒ ‒

Sage Science 0.75% Agarose Cassette for BluePippin, 4 – 20 kb High Pass

(with S1 Marker)

BLF7510 + ‒ + + + ‒ ‒ ‒ ‒ ‒ ‒

HIG

HLY

REC

OM

MEN

DED

FO

R V

AR

IAN

T D

ETEC

TIO

N

HIG

HLY

REC

OM

MEN

DED

FO

R D

E N

OVO

ASS

EMB

LY

(HIF

I, M

ICR

OB

IAL)

AN

D S

V D

ETEC

TIO

N


F. General Laboratory Equipment

WGS RNA META TARG

Vendor Product PN

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Det

ectio

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Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Benchtop Microcentrifuges Eppendorf See recommended models below

MiniSpin® Plus

Centrifuge 5425

022620100

5405000441

+ + + + + + + + + + +

Mini Microcentrifuges VWR Mini Microcentrifuge

10153-842 + + + + + + + + + + +

Plate Centrifuge VWR Plate Centrifuge

10144-214 + + + + + + + + + + +

Tube Rotators VWR Tube Rotator

10136-084 + + + + + + + + + + +


F. General Laboratory Equipment (Cont.) WGS RNA META TARG

Vendor Product PN

De

Nov

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Det

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Iso-

Seq

Sing

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Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Vortex Mixers VWR Digital Vortex Mixer

(or equivalent)

10153-842 + + + + + + + + + + +

VWR Microtube and microplate holder kit with insert retainer for vortex mixer

Q33230 + + + + + + + + + + +

Thermal Mixers Eppendorf

Eppendorf ThermoMixer® C

(or equivalent)

Provides stable incubation temperatures for SMRTbell library construction reactions

2231000667 + + + + + + + + + + +

Thermal Cyclers Bio-Rad S1000 Thermal Cycler

(or equivalent)

Provides stable incubation temperatures for SMRTbell library annealing and binding reactions Controllable heated lid preferred Compatible with 0.2 mL PCR

tubes 96-well capacity recommended

for high-throughput environments Minimum 60 minutes thermal

stability needed for 4ºC – 80ºC Overnight thermal stability

needed at 4ºC

1861096 + + + + + + + + + + +


F. General Laboratory Equipment (Cont.)

WGS RNA META TARG

Vendor Product PN

De

Nov

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i

De

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DN

A In

put

De

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Mic

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Stru

ctur

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Det

ectio

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Iso-

Seq

Sing

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Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Heat Sealers and Foil Sheets Thermo Fisher

Scientific ALPS 50 V-Manual Heat Sealer

(or equivalent)

Note that if you already have a

heat sealer, it must be capable of maintaining temperatures between 160ºC to 178ºC. Heat sealers exceeding this range may have negative impacts on sequencing results.

AB-1443A + + + + + + + + + + +

Thermo Fisher Scientific

Pierceable Sealing Foil Sheets

AB-0757 + + + + + + + + + + +

Pipettes and Tips Gilson

PIPETMAN® P2, P10, P20, P100,

P200, P1000 Pipettes (or equivalent)

F144801 F144802 F123600 F123615 F123601 F123602

+ + + + + + + + + + +

Gilson Filter Tips for PIPETMAN® P2, P10, P20, P100, P200, P1000 Pipettes

(or equivalent)

F1731031 F1733031 F1735031 F172613

+ + + + + + + + + + +

F. General Laboratory Equipment (Cont.)


WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

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Low

DN

A In

put

De

Nov

o –

Mic

robi

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Varia

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etec

tion

Stru

ctur

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aria

nt

Det

ectio

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Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Pipettes and Tips (Cont.) Rainin Wide-Orifice Pipette Tips RT LTS

200 µL FLW 960A/10 (or equivalent)

30389241 + + + + + + + + + + +

Ice Makers VWR Flake Ice Maker with Bin

(or equivalent)

89166-534 + + + + + + + + + + +

Magnetic Separator Racks Thermo Fisher

Scientific DynaMag™-2 Magnet

Separation Stand for 1.5–2 mL Microcentrifuge Tubes (or

equivalent)

12321D + + + + + + + + + + +

V & P Scientific, Inc.

Low Profile Magnetic Separation Stand for 0.2 mL, 8-Strip PCR

Tubes (or equivalent)

VP772F4-1 + + + + + + + + + + +


General Laboratory Equipment (Cont.)

WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

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Low

DN

A In

put

De

Nov

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Mic

robi

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etec

tion

Stru

ctur

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aria

nt

Det

ectio

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Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Lab Refrigerators (4ºC) VWR Standard Series Lab Refrigerator

(or equivalent)

76314-274 + + + + + + + + + + +

Lab Freezers (-20ºC) VWR Standard Series Lab Freezer

with Manual Defrost (or equivalent)

76314-276 + + + + + + + + + + +


F. General Laboratory Reagents and Consumables

WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

o –

Low

DN

A In

put

De

Nov

o –

Mic

robi

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Varia

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etec

tion

Stru

ctur

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aria

nt

Det

ectio

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Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Tubes* Eppendorf Eppendorf PCR Tubes, 0.2 mL,

PCR Clean, Colorless 0030124332 + + + + + + + + + + +

Eppendorf Eppendorf DNA LoBind Tube 0.5 mL, PCR clean

022431005 + + + + + + + + + + +


022431021 + + + + + + + + + + +


022431048 + + + + + + + + + + +

VWR Falcon™ Conical Centrifuge Tubes, PP (Polypropylene), 15 mL / 50 mL

21008-936 21008-940 + + + + + + + + + + +

* Corning® Axygen® consumables are not compatible with SMRT Sequencing and should not be used in the preparation of PCR amplicons or SMRTbell® templates.


General Laboratory Reagents and Consumables (Cont.)

WGS RNA META TARG

Vendor Product PN

De

Nov

o –

HiF

i

De

Nov

o –

Low

DN

A In

put

De

Nov

o –

Mic

robi

al

Varia

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etec

tion

Stru

ctur

al V

aria

nt

Det

ectio

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Iso-

Seq

Sing

le-C

ell

Iso-

Seq

Full-

Leng

th 1

6S

Shot

gun

Met

a-G

Am

plic

on S

eq.

No-

Am

p Se

q.

Ethyl Alcohol, Pure Sigma-Aldrich Ethyl Alcohol, Pure, 200 Proof,

ACS Reagent, ≥99.5% (or equivalent)

459844 + + + + + + + + + + +

Molecular Biology Grade Water Sigma-Aldrich Nuclease-Free Water, for Molecular

Biology (or equivalent)

W4502 + + + + + + + + + + +

Sample and Buffer Plate Accessories for Agilent Fragment Analyzer and Femto Pulse Systems

Eppendorf twin.tec® PCR Plate 96, semi-skirted, 250 µL, PCR clean,

colorless plates (Recommended Sample Plate for Agilent Fragment Analyzer & Femto Pulse Systems)

951020303 + + + + + + + + + + +

Thermo Fisher Scientific

Fisherbrand™ 96-Well DeepWell™ Polypropylene Microplates, 1 mL (Recommended Buffer Plate for

Agilent Fragment Analyzer & Femto Pulse Systems)

12-566-120 + + + + + + + + + + +

For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020, 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/o r use restrictions may pertain to your use of Pacific Biosciences products and/or third p arty products. Please refer to the applicable Pacific Biosciences Terms and Conditions of S ale and to the applicable license terms at http://www.pacificbiosciences.com/lice nses.html. Pacific Biosciences, the Pacific Biosciences logo, PacBio, S MRT, SMRTbell, Iso-Seq and Sequel are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science, Inc. NGS-go and NGSengine are trademarks of GenDx. FEMTO Pulse and Fragment Analyzer are trademarks of Agilent Technologies. All other trademarks are the sole property of their respective owners.

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Quick Reference Card - Equipment and Materials …...De Novo – HiFi Low DNA De Novo Seq – Input Sequencing De Novo Length – hotgun Meta M icrobial Variant Detection Structural

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