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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?