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Introduction to the

NextSeq System

2

Course Objectives

By the end of this course, you will be able to:

– Identify features of the NextSeq System

– Describe NextSeq Consumables

– Describe NextSeq Sequencing Technology

3

Exome, transcriptome, whole genome sequencing – in a single run

Sample size flexibility

Flow cell options (e.g. Mid or High Output flow cells) can be used to

select data output levels

Scale from a single sample to dozens of samples per run

Load & Go workflows

Integrated sample-to-results solution: streamlined

informatics on-premise or in cloud

NextSeq 500

4

High

120 GIGABASES | PE150

400M | CLUSTERS

Mid

40 GIGABASES | PE150

130M | CLUSTERS

.

One System, Two Output Modes

5

NextSeq Series Performance Parameters

Total times include cluster generation, sequencing and base calling on a NextSeq 500 System.

Install specifications are based on Illumina PhiX control library at supported cluster densities (between 170 and

230 k/mm2 clusters passing filter. Actual performance parameters may vary based on sample type, sample

quality, and clusters passing filter.

The percentage of bases > Q30 is averaged over the entire run.

8

NextSeq Sequencing Technology

9

Cluster Generation

Library pool loaded into reagent cartridge

flows through all 4 lanes of the flow cell

Hybridization and cluster generation are

automated on the NextSeq system

Approximately 5,000 molecules are included

in a cluster

Single

DNA

Library

Amplified

Clonal

Cluster

10

Two Channel Sequencing only

requires 2 images and hence all

the data from the four DNA

bases is encoded in these two

images.

This works much like Four

Channel Sequencing and uses

the same sequencing by

synthesis (SBS) method but

allows more efficient acquisition

of the data.

NextSeq 500 Imaging Cycles

Green Channel Image

Red Channel Image

11

Four Channel SBS Chemistry: GA, HiSeq, MiSeq

Collects 4 images• During each cycle,

each cluster appears

in only 1 of 4 images

Each of the 4 DNA bases

emit an intensity of a

unique wavelength

12

Two channel SBS uses 2 images

Builds template over 5 cycles

Clusters appearing in green only are T

Clusters appearing in red only are C

Clusters appearing in both images are A

Clusters not present in either green nor

red are G

Cluster intensities are plotted and bases

are called accordingly

Two Channel SBS – NextSeq Series

13

SBS Evolution – 50% Reduction in Imaging

Same library prep,

clustering and SBS

New 2-channel

imaging

14

1 2 3

Paired End

Turnaround

4

Paired End Dual Index Sequencing Utilizes 4 Sequencing Reads

Sequencing Paired End Libraries with Dual Index

Read: GAIIx, HiSeq, MiSeq

15

Index read 3

i5 Index Seq

Primer

(SBS3rev/BP13)

i5 Index

Index Read 2 (i5)

i7 Index

1 2 3

Paired End

Turnaround

4

Sequencing Paired End Libraries with Dual Index

Read: NextSeq

• Dual-indexed single reads will need to go through paired end turn around

• During run set up, do not enter cycles for read 2

Note on NextSeq Single Read Dual Index Sequencing:

16

NextSeq Hardware

18

System enhancements: Optics

Power of high throughput appsSize and affordability of a desktop sequencer.

6xMiSeq imaging

capability

1/3Size of a HiSeq

1/3Capital cost

of a HiSeq

6 parallel miniaturized,

solid-state optics modules

20

System enhancements: Fluidics

No TubesEasily

swapped out

ReducedVolumes

LowerMaintenance

21

CARTRIDGE FORMAT | AUTOMATED WASH | SELF-CLEANING

NextSeq Consumables

22

NextSeq 500/550 Consumables

23

NextSeq 500/550 Kit Contents

NextSeq 500/550 Kit Sizes

75 cycles 93 cycles of sequencing

150 cycles 168 cycles of sequencing

300 cycles 318 cycles of sequencing

• Store at -15°C to -25°C

Reagent Cartridge

• Store at RT

Buffer Cartridge

• Store at -15°C to -25°C

• HT1: hybridization buffer

Accessory Box

• Store at 2°C to 8°C

Flow Cell

24

Each kit type contains a compatible flow cell and reagent cartridge

The buffer cartridge is universal

Always check the label markings when you prepare consumables

for a run

NextSeq 500/550 Kit Options

NextSeq 500/550 kits are available in two output types:

NextSeq 500/550 High Output Kit

Options: 75 cycles, 150 cycles, 300 cycles

NextSeq 500/550 Mid Output Kit

Options: 150 cycles, 300 cycles

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Load & Go Flow cell

Color-Coded Icons

At a glance ID of kit

compatibility

Plastic Cartridge

Protects against

drops

Handle

For tactile feedback

Ships Dry

HIG

H

26

Flow Cell Design

Dual-surface enabled

4 lane format

1 sample/pool is distributed across all 4 lanes

Images in lane pairs

– Lane Pair A: Lanes 1 and 3

– Lane Pair B: Lanes 2 and 4

Reagent flow

– Lane Pair A: top to bottom

– Lane Pair B: bottom to top

Flow Cell Cartridge

Lanes Pair A Lane Pair B

Reagent Flow:

Lane Pair A

Reagent Flow:

Lane Pair B

27

Flow Cell Format

Camera

Section

L 4

L 3L 2

Lane 1

Tile#

Swath1Swath2

Swath3

Rear

Front

Nomenclature: 2_1 2 1 04

Tile

Section

Swath

Top / Bottom

Lane

Example: s_1_21304_red.tif = Lane 1,

Bottom Surface, Swath 1, Camera 3, Tile 4

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Preloaded single-use reagent

cartridge

– Contains all reagents required

for cluster generation,

sequencing and paired-end

chemistry

Single library or pool of indexed

libraries can be loaded into

cartridge

Contains one wash reservoir for

every reagent reservoir

NextSeq 500/550 Reagent Cartridge

Wash reservoir

29

The NextSeq buffer cartridge is

a single-use consumable

containing three reservoirs filled

with buffers and wash solution

Contents of the buffer cartridge

are sufficient for one sequencing

run

NextSeq 500/550 Buffer Cartridge

Buffer Position Reagent Description

1 BB4: Sequencing buffer

2 BB3: Wash solution

3 BB2: Incorporation buffer

30

Spent Reagents Tray

When removing or loading the spent reagents tray, be sure to place one

hand underneath the tray for support

Spent Reagents Tray

31

NextSeq Software

32

NCS guides user through

all steps to load

consumables and start run

Automatic wash is

performed after every

sequencing run

NextSeq Control Software (NCS)

33

NextSeq Data Analysis

• Performed by NCS + RTA2

• On-instrument computer performs all primary analysis

Primary Analysis

• Run folder is sent to a network drive and/or BaseSpace

• Large BCL files: one per lane and cycle

• Data is compressed: zipped bcl files and binned Qscores

Run Data Output

• Performed in BaseSpace or in Linux using bcl2fastq 2.0+

• No fastq conversion occurs on the instrument

Demultiplexing and bcl to fastq conversion

34

BaseSpace Integration

35

Before run: library information

and run parameters can be

entered in BaseSpace

During run set-up: runs set up in

BaseSpace appear on NCS

interface for selection

During sequencing run: data

can be streamed in real time to

BaseSpace

– BaseSpace is available on the

cloud or through BaseSpace

Onsite

NextSeq BaseSpace Integration

36

NextSeq Workflows

37

High-Output

Flow Cell Configuration

Mid-Output

Flow Cell Configuration

NextSeq Workflow Options

Gene Expression Profiling

> 10 M Reads

2 x 75 bp

N/A40 Samples

12 Hours

mRNA-Seq

> 40 M Reads

2 x 75 bp

3 Samples

15 Hours10 Samples

18 Hours

Enrichment Panel

12 Mb Region

>20x Coverage

2 x 150 bp

20 Samples

26 Hours N/A

Whole-Exome

>90% at > 10x coverage

2 x 150 bp

3 Samples

15 Hours9 Samples

29 Hours

Human Whole Genome

3 GB Genome

> 30x Coverage

2 x 150 bp

1 Sample

29 Hours

38

NextSeq Sample-to-Answer Workflow

Prep≥15 minutes

hands-on

≥ 1.5 HOURS

Sequence20 minutes

hands-on

10-30HOURS

Analyzefully automated,

BaseSpace

up to 24HOURS/SAMPLE

Sharesecure, unlimited

storage

INSTANTLY

40

Questions?