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NextSeq Sequencing

Workflow Overview

2

Course Objectives

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

– List the NextSeq System Sequencing workflow steps

– Prepare a NextSeq run in BaseSpace

– Prepare and load NextSeq 500/550 reagents

– Start a sequencing run using NextSeq Control Software

– Discuss NextSeq wash options

3

NextSeq Sample-to-Answer Workflow

Choose Instrument Configuration

BaseSpace

Configuration

(Cloud or

Onsite)

Stand-alone

Configuration

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

4

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

Enter run parameters

in BaseSpace

Prepare libraries

5

BaseSpace Prep Tab

What is it

– You can prepare NextSeq runs through the BaseSpace Prep tab, which

organizes samples, libraries, pools, and run in a single environment.

When to use it

– Use this if you want to prepare a sequencing run on a NextSeq instrument

and have the data stream seamlessly to BaseSpace.

– Do not use it to prepare sequencing runs for other instruments or if you

prefer to start a run straight from the NextSeq instrument.

Why to use it

– Preparing a run in the Prep tab moves the data and analysis seamlessly to

BaseSpace.

6

BaseSpace Prep Tab

Enter run parameters

in BaseSpace

Prepare Libraries

Enter run parameters in

BaseSpace

Create Biological Samples

7

BaseSpace Prep Tab

Enter run parameters

in BaseSpace

Prepare Libraries

Enter run parameters in

BaseSpace

Assign Indexes* to Libraries

*If index N705 is chosen, use at least 1 more i7 index in your pool

8

BaseSpace Prep Tab: Custom Library Prep

feature

Enter run parameters

in BaseSpace

Prepare Libraries

Enter run parameters in

BaseSpace

To define a new custom library prep (including indices): 1. Select the biological samples and click Prep Libraries

2. At the Prep Libraries page, select the new + Custom Library Prep

Kit under the Library Prep kits drop down list.

9

BaseSpace Prep Tab: Custom Library Prep

feature

Enter run parameters

in BaseSpace

Prepare Libraries

Enter run parameters in

BaseSpace

A Custom Library Prep Kit definition page will open

10

BaseSpace Prep Tab

Enter run parameters

in BaseSpace

Prepare Libraries

Enter run parameters in

BaseSpace

Add libraries to pool

11

BaseSpace Prep Tab

Enter run parameters

in BaseSpace

Prepare Libraries

Enter run parameters in

BaseSpace

Plan Run

12

Library Preparation

Enter run parameters

in BaseSpace

Prepare Libraries

The aim of the library prep step is to obtain nucleic

acid fragments with adapters attached on both ends

Library Prep is Critical for Successful Sequencing

Dual Index Library shown

13

Library Preparation

Other Sequencing Options*:

– TruSeq ChIP-Seq

– Nextera Mate Pair

– Nextera XT DNA library prep

– TruSeq Synthetic Long Reads

*NextSeq Extended Secondary

Analysis options are not yet

available for these kits

For DNA Sequencing:

• TruSeq Nano DNA

• TruSeq DNA PCR-Free

• Nextera DNA

• TruSeq DNA Methylation

For Enrichment Sequencing:

• Nextera Rapid Capture Enrichment

• Exome

• Expanded Exome

• TruSight Rapid Capture and TruSight One

For Amplicon Sequencing:

• TruSeq Custom Amplicon

• TruSeq Amplicon Cancer Panel

• TruSight Myeloid

• TruSight Tumor

For RNA Sequencing:

• TruSeq Stranded mRNA

• TruSeq Stranded Total RNA

• TruSeq RNA v2

• TruSeq RNA Access

• Targeted RNA Expression

• Small RNA

14

Not Sure Which Kit to Use?Use the Library Prep Kit Selector

http://www.illumina.com/library-prep-array-kit-selector.html

15

Optional: Import Libraries in Prep Tab

Enter run parameters

in BaseSpace

Prepare Libraries

This feature lets you start with prepared libraries, eliminating

the need to first enter biological samples.

16

Optional: Import Libraries in Prep Tab

Enter run parameters

in BaseSpace

Prepare Libraries

1. Once on the Import Sample Libraries Page, click template

to download the file template

2. Click the Choose.csv button to select a file for upload.

17

How to Start a NextSeq Run

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

Denature and dilute

Prepare reagent

cartridge

Prepare flow cell

Load libraries into

reagent cartridge

Start run in NCS

18

Library Preparation

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Start run in NCS

Denature and dilute your sample

– Target cluster densities (170 – 230 K clusters/mm2)

– Denature and dilution for the NextSeq requires 200 mM

Tris-HCl, pH7, a user-supplied consumable

200 mM Tris-HCl, pH7 ensures NaOH is fully hydrolyzed in the

final solution

Denature and dilute PhiX 1.8 pM*

Mix sample library and PhiX control to result in a 1% PhiX

control volume ratio

A total of 1.3 mL* of denatured and diluted sample is

needed for loading into the reagent cartridge

*Load 3 mL at 3pM concentration if using NCS v1.2, or earlier

Denature and dilute

Prepare flow cell

19

Thaw cartridge in RT water bath for 60 min to 90

min

– Do not submerge above lower lid edge

– Ensure all reagents are fully thawed

Manually invert cartridge 5X

Tap cartridge on hard surface to remove bubbles

and dislodge water from cartridge base

Dry the base of the cartridge

NextSeq 500/550 Reagent Cartridge Preparation

Water Line

Prepare reagent cartridge

Denature and Dilute

Load libraries into

reagent cartridge

Start run in NCS

Prepare flow cell

21

NextSeq 500/550 Reagent Cartridge v2 Overview

Position(s) Reagent

Description

1-5 Wash Reservoir

6 Denaturation

reagent

7-9 Reserved for

custom primers

10 Library loading

reservoir

11 0.12% Bleach

12-17 Wash Reservoir

18 Conditioning

reagent

19 Resynthesis mix

20 Read 1

sequencing primer

Position(s) Reagent

Description

21 Read 2

sequencing primer

22 Index sequencing

primers

23 Read 1

linearization mix

24 Read 2

linearization mix

25-28 Wash Reservoir

29 Cleavage mix

30 Scan mix

31 Incorporation mix

32 Amplification mix

24

A. Custom Read 1 primer

B. Custom Read 2 primer

C. v1: Custom Index 1 primer

v2: Custom Index 1 and Index 2 primers

Custom Primers

Load Custom Primers

Denature and Dilute

Load libraries into

reagent cartridge

Start run in NCS

Prepare flow cell

Prepare reagent

cartridge

Note: Custom Index 2 primer is

not supported with v1 reagents.

25

Leave the flow cell package at room temperature

for 30 minutes before use

Remove the foil packaging and then remove the

flow cell cartridge from the plastic clamshell casing

Clean the glass surface of the flow cell using an

alcohol wipe

Dry the glass with a lint-free tissue or lens paper

Prepare the Flow Cell

Prepare flow cell

Denature and Dilute

Load libraries into

reagent cartridge

Start run in NCS

Prepare reagent

cartridge

Note: After the foil packaging has been opened,

use the flow cell within the next 12 hours.

26

Inspect Flow Cell

Inspect the four metal spring clips on

both the top and back sides of the

flow cell

On the back side of the flow cell,

visually inspect the flow cell ports,

gaskets, and carrier plate

Make sure that the black

carrier plate is flat and

secure under the four

spring clips

Make sure the port gaskets

are seated flat against the

surface.

v

Make sure that the ports are

free of obstructions.

27

Load sample in highlighted position on reagent

cartridge

– Pierce foil

– Add sample by pipetting to bottom of well

For NCS v1.3, or later – Load 1.3 ml library

For NCS v1.2, or earlier – Load 3 ml library

– Avoid air bubbles

Load Libraries into Cartridge

Load libraries into reagent

cartridge

Denature and Dilute

Prepare reagent

cartridge

Start run in NCS

Prepare flow cell

28

Start the Run in NCS

Start run in NCS

Touch screen Graphical User Interface

Oversized buttons make on-screen navigation easy

Log into BaseSpace to pull in run set-up parameters

Starting a run only takes a few clicks

Denature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Prepare flow cell

29

NextSeq Kit Versions and Software

Start run in NCS

NextSeq 500/550 Kit v2 requires NCS 1.4+

If using NextSeq Kit v1, do not upgrade beyond NCS 1.3

Important Customer Information package insert will ship

with NextSeq 500/550 Kit v2

Denature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Prepare flow cell

30

NextSeq Control Software and Radio Frequency Identification (RFID)

Each consumable loaded into NextSeq

contains a unique Radio Frequency ID, or

RFID

The RFID is recognized by the NCS, allowing the

software to automatically populate the barcode numbers of each consumableStart run in NCS

Denature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Prepare flow cell

Jump to Post-Run

Procedures

Note: A brief wait after loading each

NextSeq consumable is normal.

31

Stand-alone mode

32

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

Prepare libraries

33

Library Preparation

Prepare Libraries

The aim of the sample prep step is to obtain nucleic

acid fragments with adapters attached on both ends

Library Prep is Critical for Successful Sequencing

Dual Index Library shown

34

Library Preparation

Other Sequencing Options*:

– TruSeq ChIP-Seq

– Nextera Mate Pair

– Nextera XT DNA library prep

– TruSeq Synthetic Long Reads

*NextSeq Extended Secondary

Analysis options are not yet

available for these kits

For DNA Sequencing:

• TruSeq Nano DNA

• TruSeq DNA PCR-Free

• Nextera DNA

• TruSeq DNA Methylation

For Enrichment Sequencing:

• Nextera Rapid Capture Enrichment

• Exome

• Expanded Exome

• TruSight Rapid Capture and TruSight One

For Amplicon Sequencing:

• TruSeq Custom Amplicon

• TruSeq Amplicon Cancer Panel

• TruSight Myeloid

• TruSight Tumor

For RNA Sequencing:

• TruSeq Stranded mRNA

• TruSeq Stranded Total RNA

• TruSeq RNA v2

• TruSeq RNA Access

• Targeted RNA Expression

• Small RNA

35

Not Sure Which Kit to Use?Use the Library Prep Kit Selector

http://www.illumina.com/library-prep-array-kit-selector.html

36

How to Start a NextSeq Run

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

Denature and dilute

Prepare reagent

cartridge

Prepare flow cell

Load libraries into

reagent cartridge

Start run in NCS

37

Library Preparation

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Start run in NCS

Denature and dilute your sample

– Target cluster densities (170 – 230 K clusters/mm2)

– Denature and dilution for the NextSeq requires 200 mM

Tris-HCl, pH7, a user-supplied consumable

200 mM Tris-HCl, pH7 ensures NaOH is fully hydrolyzed in the

final solution

Denature and dilute PhiX 1.8 pM*

Mix sample library and PhiX control to result in a 1% PhiX

control volume ratio

A total of 1.3 mL* of denatured and diluted sample is

needed for loading into the reagent cartridge

*Load 3 mL at 3pM concentration if using NCS v1.2, or earlier

Denature and dilute

Prepare flow cell

38

Thaw cartridge in RT water bath for 60 min to 90

min

– Do not submerge above lower lid edge

– Ensure all reagents are fully thawed

Manually invert cartridge 5X

Tap cartridge on hard surface to remove bubbles

and dislodge water from cartridge base

Dry the base of the cartridge

NextSeq 500/550 Reagent Cartridge Preparation

Water Line

Prepare reagent cartridge

Denature and Dilute

Load libraries into

reagent cartridge

Start run in NCS

Prepare flow cell

40

NextSeq 500/550 Reagent Cartridge v2 Overview

Position(s) Reagent

Description

1-5 Wash Reservoir

6 Denaturation

reagent

7-9 Reserved for

custom primers

10 Library loading

reservoir

11 0.12% Bleach

12-17 Wash Reservoir

18 Conditioning

reagent

19 Resynthesis mix

20 Read 1

sequencing primer

Position(s) Reagent

Description

21 Read 2

sequencing primer

22 Index sequencing

primers

23 Read 1

linearization mix

24 Read 2

linearization mix

25-28 Wash Reservoir

29 Cleavage mix

30 Scan mix

31 Incorporation mix

32 Amplification mix

43

A. Custom Read 1 primer

B. Custom Read 2 primer

C. v1: Custom Index 1 primer

v2: Custom Index 1 and Index 2 primers

Custom Primers

Load Custom Primers

Denature and Dilute

Load libraries into

reagent cartridge

Start run in NCS

Prepare flow cell

Prepare reagent

cartridge

Note: Custom Index 2 primer is

not supported with v1 reagents.

44

Leave the flow cell package at room temperature

for 30 minutes before use

Remove the foil packaging and then remove the

flow cell cartridge from the plastic clamshell casing

Clean the glass surface of the flow cell using an

alcohol wipe

Dry the glass with a lint-free tissue or lens paper

Prepare the Flow Cell

Prepare flow cell

Denature and Dilute

Load libraries into

reagent cartridge

Start run in NCS

Prepare reagent

cartridge

Note: After the foil packaging has been opened,

use the flow cell within the next 12 hours.

45

Inspect Flow Cell

Inspect the four metal spring clips on

both the top and back sides of the

flow cell

On the back side of the flow cell,

visually inspect the flow cell ports,

gaskets, and carrier plate.

Make sure that the black

carrier plate is flat and

secure under the four

spring clips

Make sure the port gaskets

are seated flat against the

surface.

v

Make sure that the ports are

free of obstructions.

46

Load sample in highlighted position on reagent

cartridge

– Pierce foil

– Add sample by pipetting to bottom of well

For NCS v1.3, or later – Load 1.3 ml library

For NCS v1.2, or earlier – Load 3 ml library

– Avoid air bubbles

Load Libraries into Cartridge

Load libraries into reagent

cartridge

Denature and Dilute

Prepare reagent

cartridge

Start run in NCS

Prepare flow cell

47

NextSeq Kit Versions and Software

Start run in NCS

NextSeq 500/550 Kit v2 requires NCS 1.4+

If using NextSeq Kit v1, do not upgrade beyond NCS 1.3

Important Customer Information package insert will ship

with NextSeq 500/550 Kit v2

Denature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Prepare flow cell

48

Start the Run in NCS

Start run in NCS

Touch screen Graphical User Interface

Oversized buttons make on-screen navigation easy

Simple Run SetupDenature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Prepare flow cell

\\your-network\drive

Save run to a network drive

<< your-network\drive

49

Start the Run in NCS

Start run in NCS

Start run after pre-run checks passDenature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Prepare flow cell

50

Run Monitoring Option in NCS 1.4+

Start run in NCS

Optional box to select BaseSpace Run MonitoringDenature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Load fresh dilution of

NaOCl

Prepare flow cell

\\your-network\drive

51

NextSeq Control Software and Radio Frequency Identification (RFID)

Each consumable loaded into NextSeq

contains a unique Radio Frequency ID, or

RFID

The RFID is recognized by the NCS, allowing the

software to automatically populate the barcode numbers of each consumableStart run in NCS

Denature and Dilute

Prepare reagent

cartridge

Load libraries into

reagent cartridge

Prepare flow cell

Note: A brief wait after loading each

NextSeq consumable is normal.

52

Post-Run Procedures

53

Post run wash

Removable reservoir

Manual wash

Instrument Disk Space

Idling the instrument

Shutting down the instrument

NextSeq Post-Run Procedures

54

An automatic wash is

performed after every

sequencing run

Leave the used flow cell,

reagent cartridge and

buffer cartridge on the

instrument when the

wash is complete

NextSeq Post-Run Wash

Expected Wash

Deliveries

Approximate Run

Time

17.25 ml 90 minutes

55

Position 6 of the reagent

cartridge includes a

denaturation reagent (LDR) that

contains formamide

The removable reservoir in

position 6 allows for safe

disposal of any unused LDR

after the sequencing run

Removable Reservoir

A. Protective removable tab

B. Position 6

56

Perform manual

instrument wash if

instrument has been

idle for 14 days or more

– Load

– Check

– Wash

Always use a wash

solution of laboratory-

grade water mixed with

Tween 20 (0.05%

Tween 20 final

concentration)

Manual Instrument Wash

57

Manual Wash options (NCS 1.4+)

Quick Wash

• Duration: 20 minutes

• Flushes system with wash buffer

• Use when instrument has been idle for 14 days or more or when restarting after instrument shut down

Manual Post-Run Wash

• Duration: 90 minutes

• Flushes system with wash buffer plus 0.12% NaOCl added to reagent wash cartridge

• Use if run was interrupted and automatic post-run wash was not performed

59

Prepare Buffer Wash Tray

– Load 125 mL of fresh 0.05%

Tween 20 wash solution into

middle reservoir of buffer

wash tray only

Add NaOCl (bleach) into

reservoir #28 of reagent wash

tray only if performing:

– Manual post-run wash (NCS

1.4+)

Manual Wash Trays

60

Leave the used flow

cell, wash tray and

spent reagents tray on

the instrument when

the wash is complete

Manual Instrument Wash

61

The NextSeq system only has

enough disk storage to hold

data from one complete run

Best practice is to save

sequencing run to BaseSpace

or a network drive

Ensure that the any run data

from has been cleared from the

instrument hard drive

On-instrument Disk Space

62

Safely shut down the instrument

from the Manage Instrument

button

Restart the instrument

– Any time that you turn off the

instrument, wait a minimum of

60 seconds before switching

back on

– Toggle the power switch back

to the ON position

NextSeq System Powering: Shut Down and

Restart

63

Best Practices

64

RFID will take several seconds to register, please be patient

Empty the spent reagents cartridge before each run

Ensure that dilution of bleach/NaOCl is made from a FRESH stock of bleach/NaOCl

Keep wash cartridge clean, keep inverted when not in use

Leave the used flow cell, reagent cartridge and buffer cartridge on the instrument until your next run

Save run data to a network or BaseSpace

It is best to leave the NextSeq 500 instrument on at all times

NextSeq Best Practices

66

NextSeq Analysis Options

• BWA Whole Genome Sequencing

• Isaac Whole Genome Sequencing

• Tumor NormalDNA Sequencing

• TopHat Alignment and Cufflinks DE

• RNA Express

• Small RNARNA Sequencing

• Designed to detect somatic variants from a tumor and matched normal sample pairEnrichment

• TruSeq Amplicon

• Amplicon DS

• TruSeq Targeted RNAAmplicon

• 16S MetagenomicsMetagenomics

• TruSeq Long Read Assembly

• TruSeq Phasing AnalysisAssembly and Phasing

• MethylSeqMethylation

• A stand-alone converter of BCL files to generate FASTQ files for universal use Bcl2FastQ2

BaseS

pace

Only

69

Share NextSeq Run

70

Questions?