Introduction to the

NextSeq 500 System

Abhishek Singh
Field Applications Scientist

PREMAS Lifesciences

© 2013 Illumina, Inc. All rights reserved.

Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium,
iSelect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks
of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
Course Objectives

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

– Identify features of the NextSeq 500 System
– Describe NextSeq 500 Consumables
– Describe NextSeq 500 Sequencing Technology

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NextSeq 500 Overview

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NextSeq 500

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

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One System, Two Output Modes

High Mid
120 GIGABASES | PE150 40 GIGABASES | PE150
400M | CLUSTERS 130M | CLUSTERS

.
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NextSeq 500 Performance Parameters

Read Length Output

Flow Cell Configuration Run Time Data Quality
(bp) (Gb)

2 x 150 100-120 30 hours

Hi Output Flow Cell
Up to 400 M single reads 2 x 75 50-60 18 hours
Up to 800 M paired-end reads  75% > Q30 at 2 x 150 bp
1 x 75 25-30 12 hours  80% > Q30 at 2 x 75 bp

Mid Output Flow Cell 2 x 150 32.5-39 26 hours

Up to 130 M single reads
Up to 260 M paired-end reads 2 x 75 16.25-19.5 15 hours

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.

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NextSeq 500 Sequencing Technology

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Cluster Generation

Single Amplified
DNA Clonal
Library Cluster

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

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NextSeq 500 Imaging Cycles

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 Green Red
sequencing by synthesis (SBS) Channel Channel
method but allows more efficient Image Image
acquisition of the data.

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Four Channel SBS Chemistry: GA, HiSeq, MiSeq

Each of the 4 DNA bases

emit an intensity of a
unique wavelength

Collects 4 images
• During each cycle,
each cluster appears
in only 1 of 4 images

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Two Channel SBS – NextSeq 500

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

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SBS Evolution – 50% Reduction in Imaging

Same library prep,

clustering and SBS

New 2-channel
imaging

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Sequencing Paired End Libraries with Dual Index Read:
GAIIx, HiSeq, MiSeq

1 2 3 4

Paired End
Turnaround

Paired End Dual Index Sequencing Utilizes 4 Sequencing Reads

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Sequencing Paired End Libraries with Dual Index Read:
NextSeq
Index Read 2 (i5)

1 2 3 4
i7 Index

Paired End
Turnaround
Index read 3

i5 Index Seq
Primer
(SBS3rev/BP13)
i5 Index

Note on NextSeq Single Read Dual Index Sequencing:

• Dual-indexed single reads will need to go through paired end turn around
• During run set up, do not enter cycles for read 2

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NextSeq Hardware

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NextSeq Optics

CONSUMER OPTICS | SOLID STATE | NO ALIGNMENT

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System enhancements: Optics

Power of high throughput apps

Size and affordability of a desktop sequencer.

6x 1/3 1/3
MiSeq imaging Capital cost
Size of a HiSeq
capability of a HiSeq

6 parallel miniaturized,
solid-state optics modules

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NextSeq Fluidics

INTEGRATED FLUIDICS MANIFOLD | MINIMIZED TUBING

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System enhancements: Fluidics

No Tubes Reduced Lower

Easily
Volumes Maintenance
swapped out

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NextSeq Consumables

CARTRIDGE FORMAT | AUTOMATED WASH | SELF-CLEANING

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NextSeq 500 Consumables

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NextSeq 500 Kit Contents

NextSeq 500 Kit Sizes Reagent Cartridge

75 cycles 92 cycles of sequencing • Store at -15°C to -25°C
150 cycles 168 cycles of sequencing
300 cycles 318 cycles of sequencing Buffer Cartridge

• Store at RT

Flow Cell

• Store at 2°C to 8°C

Accessory Box

• Store at -15°C to -25°C

• BP13: NextSeq Dual
Indexing Primers
• HT1: hybridization buffer

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NextSeq 500 Kit Options

NextSeq 500 kits are available in two output

types:

NextSeq 500 High Output Kit

Options: 75 cycles, 150 cycles, 300 cycles

NextSeq 500 Mid Output Kit

Options: 150 cycles, 300 cycles

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

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Load & Go Flow cell

Color-Coded Icons

HIGH
At a glance ID of kit
compatibility

Ships Dry

Handle
For tactile feedback

Plastic Cartridge
Protects against
drops

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Flow Cell Design
Lanes Pair A Lane Pair B

Dual-surface enabled
4 lane format
1 sample/pool is distributed across all 4 lanes
Images in lane pairs Reagent Flow:
Lane Pair B
– 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 Reagent Flow:
Lane Pair A

Flow Cell Cartridge

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 Rear
Flow Cell Format
Tile#

Camera
Section

Swath1 Lane 1 L4
Swath3
Swath2
L2 L3
Top / Bottom Section Front

Nomenclature: 2_1 2 1 04 Example: s_1_21504_red.tif = Lane 1,

Bottom Surface, Swath 1, Camera 5, Tile 4
Lane Swath Tile
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NextSeq 500 Reagent Cartridge

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

Wash reservoir

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NextSeq 500 Buffer Cartridge

The NextSeq 500 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

Buffer Position Reagent Description

1 BB4: Sequencing buffer

2 BB3: Wash solution

3 BB2: Incorporation buffer

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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

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NextSeq Software

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NextSeq Control Software (NCS)

NCS guides user through all

steps to load consumables
and start run
Automatic wash is performed
after every sequencing run

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NextSeq Data Analysis

Primary Analysis

• Performed by NCS + RTA2

• On-instrument computer performs all primary analysis

Run Data Output

• 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

Demultiplexing and bcl to fastq conversion

• Performed in BaseSpace or in Linux using bcl2fastq 2.0

• No fastq conversion occurs on the instrument

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BaseSpace Integration

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NextSeq 500 BaseSpace Integration

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

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NextSeq 500 Workflows

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NextSeq 500 Workflow Options
Mid-Output High-Output
Flow Cell Configuration Flow Cell Configuration

Gene Expression Profiling

40 Samples
> 10 M Reads N/A
12 Hours
2 x 75 bp

mRNA-Seq
3 Samples 10 Samples
> 40 M Reads
15 Hours 18 Hours
2 x 75 bp

Enrichment Panel
12 Mb Region 20 Samples
>20x Coverage 26 Hours N/A
2 x 150 bp

Whole-Exome
3 Samples 9 Samples
>90% at > 10x coverage
15 Hours 29 Hours
2 x 150 bp

Human Whole Genome

3 GB Genome 1 Sample
> 30x Coverage 29 Hours
2 x 150 bp

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NextSeq 500 Sample-to-Answer Workflow

Sequence Analyze Share

Prep fully automated,
20 minutes secure, unlimited
≥15 minutes BaseSpace
hands-on storage
hands-on

≥ 1.5 HOURS 10-30 up to 24

HOURS INSTANTLY
HOURS/SAMPLE

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Questions?

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