Nextera DNA Library Preparation for NGS Express · Application Guide- Nextera DNA Library...

Nextera DNA Library Preparation

for NGS Express

Application Guide

PerkinElmer 2200 Warrenville Road

Downers Grove, IL 60515

[email protected]

Document Number 8860050

Revision A

Copyright ©2012, PerkinElmer, Inc.

All Rights Reserved

Application Guide- Nextera DNA Library Preparation on the NGS Express

PerkinElmer 2

Trademark Acknowledgement

is a registered trademark of PerkinElmer, Inc.

VersaTip™ is a trademark of PerkinElmer, Inc.

JANUS® is a registered trademark of PerkinElmer, Inc.

WinPrep® is a registered trademark of PerkinElmer, Inc.

Nextera, MiSeq, and illumina are trademarks or registered trademarks of Illumina, Inc.

Agencourt ® and AMPure® are registered trademarks of Beckman Coulter Genomics, Inc.


PerkinElmer 3

TableofContentsIntroduction .................................................................................................................................................. 4

Workflow Overview .................................................................................................................................. 4

Variations from the published protocol ................................................................................................ 5

Workflow Charts ................................................................................................................................... 7

System Setup........................................................................................................................................... 12

Materials List ....................................................................................................................................... 12

Assembling the Versa Mover .............................................................................................................. 13

Pre‐Run Instrument Checklist ............................................................................................................. 15

Running the Nextera DNA Sample Prep Method on NGS Express .............................................................. 16

Experimental Planning ............................................................................................................................ 16

Input Files ............................................................................................................................................ 16

Reagent and Sample Preparation ....................................................................................................... 18

Instrument Preparation ...................................................................................................................... 22

Running the Automated Nextera Protocols ............................................................................................ 23

Protocol 1‐ Tagmentation and Clean‐Up ............................................................................................ 23

Protocol 2‐ PCR Setup with Dual Indexing .......................................................................................... 28

Amplify the Samples ........................................................................................................................... 32

Protocol 3‐ Post‐PCR Clean‐Up ........................................................................................................... 33

Analyze Yield and Quality of the Libraries .......................................................................................... 36

Protocol 4‐ Normalization and Pooling ............................................................................................... 37

Advanced Users Guide ................................................................................................................................ 41

Custom Labware ..................................................................................................................................... 41

Versa Mover Information.................................................................................................................... 42

Protocol Logic Elements .......................................................................................................................... 42

Appendix ..................................................................................................................................................... 47

Labware & Consumables Ordering Information ..................................................................................... 47

List of Files Installed/Modified by the Nextera Option Installer ............................................................. 48


PerkinElmer 4

IntroductionThe NGS Express system is a compact, easy‐to‐use liquid handling system that automates preparation of

1‐24 samples for next‐generation DNA sequencing (NGS). The Nextera Option for NGS Express is an add‐

on package of software, hardware, and labware that enables preparation of up to 24 libraries using the

Nextera DNA Sample Preparation Kit.

Four field‐tested protocols for automating steps in the Nextera DNA Sample Prep protocol

Adapter plate for using 96‐well V‐bottom plates on the Inheco unit

Start‐up quantities of consumable labware (plates, tubes, and tips)

This automated protocol is based on the Nextera DNA Sample Preparation Guide published by Illumina

(Document Part # 15027987 Rev. A).

WorkflowOverview

Figure 1. Workflow chart showing Nextera DNA sample preparation on the NGS Express. Off‐deck steps are indicated by the

grey triangles.

AutomatedprotocolsThe automated NGS Express method for performing Nextera DNA Sample Preparation is divided into

four protocols. These protocols are illustrated as workflow charts later in this chapter.

1. Tagmentation and Cleanup. This method prepares the tagmentation reactions, incubates the

reactions on the deck, and then purifies the tagmented DNA via a double‐sided SPRI bead

procedure.

2. PCR Setup with Dual Indexing. This protocol pipets master mix, primer cocktail, and index

primers into the reactions. Reactions can be set up in either a full‐skirt PCR plate or PCR tubes.

Pipetting of index adapters is determined by reading the index selections from the Illumina

Sample Sheet, which can be generated by the Illumina Experiment Manager software.

3. Post‐PCR Cleanup. After amplification, the NGS Express will purify the libraries by SPRI (solid‐

phase reversible immobilization).

4. Normalization and Pooling. This protocol will normalize the libraries to 4nM based on the yield

(measured DNA concentration) data provided in a csv file. Optionally, equal volumes of the

normalized libraries can be pooled for multiplexed sequencing.


PerkinElmer 5

VariationsfromthepublishedprotocolThe process of automating sample preparation protocols sometimes requires that specific methods

for accomplishing certain steps be changed. The NGS Express method for Nextera DNA Sample

Preparation varies from the published Illumina manual method in a few ways:

On‐Deck Tagmentation. To provide more walk‐away time from the instrument, the tagmentation

reaction conditions were optimized to be performed on the deck of the NGS Express. Time is

increased from 5 minutes to 10 minutes and the temperature locator is set to 58°C. As shown in

Figure 2 below, equivalent results between the on‐deck and thermocycler incubation conditions are

obtained.

Figure 2. Comparison of on‐deck and in‐machine tagmentation conditions. Analysis of size distribution on a LabChip GX with

High Sensitivity chip show nearly identical size distributions of the two samples.

Use of SPRI instead of spin filter for tagmentation cleanup. To decrease manual intervention, a

magnetic bead‐based method for purifying the tagmentation reactions was implemented. In order

to dissociate the tagmentase enzyme from the DNA, guanidine HCl is added to a net concentration

of 4 M. Two sequential additions of AMPure bead reagent are effective at purifying the DNA from


PerkinElmer 6

the rest of the tagmentation reaction components, as shown in Figure 3.

Figure 3. Efficiency of guanidine for tagmentation cleanup.

Bead Drying Conditions. In order to speed up the drying process and insure complete bead

dryness, washed beads are dried at 55°C for 7 minutes.

Mixing Conditions. All bead mixing is performed by pipetting. Twenty aspirate/dispense cycles are

performed whenever adding AMPure reagent to samples or resuspending beads.

ApproximateProtocolRunTimesProtocol Time for 4 Samples Time for 24 Samples

Tagmentation and CleanUp 1 hour 45 minutes 2 hours 45 minutes

PCR Setup with Dual Indexing 5 min 24 min

Post‐PCR Cleanup 50 min 1 h 45 min

Normalization and Pooling 3 min 12 min


PerkinElmer 7

WorkflowCharts

Figure 4. Workflow chart for on‐deck tagmentation and stopping the reaction. Protocol 1 covers these steps as well as those

on the next page.


PerkinElmer 8

Figure 5. Workflow chart for post‐tagmentation cleanup using a double‐sided SPRI procedure. These steps, as well as those

on the previous page, are performed in Protocol 1.


PerkinElmer 9

Figure 6. Workflow chart for PCR setup, showing worklist generation from the customer‐provided Illumina Sample Sheet.

The steps outlined in tis figure are performed by Protocol 2.


PerkinElmer 10

Figure 7. Workflow chart for post‐PCR cleanup via a single SPRI magnetic bead method. The steps shown above are

performed by Protocol 3.


PerkinElmer 11

Figure 8. Workflow chart for library normalization and pooling (Protocol 4), showing user choices and inputs. By default,

samples are normalized to 4 nM. Normalization and/or pooling can be performed using this protocol.


PerkinElmer 12

SystemSetup

MaterialsListIncluded in the Option Package:

Software CD

Adapter Tile, 96‐well V‐bottom (for Inheco)

Disposable Tip‐ 25 µL, Clear, Sterile Filter Tips

Disposable Tip‐ 175 µL, Clear, Sterile Filter Tips

StorPlate, polypropylene, V‐bottom, 450 µL capacity

Microtubes, screw cap, 0.5 mL

Microtubes, screw cap, 2.0 mL

Hard‐Shell PCR plates, 96‐well

Eppendorf IsoRack (included with the NGS Express System)

Reagents Required but not Supplied:

Item Vendor Part No.

Nextera DNA Sample Preparation Kit (24 samples) Illumina FC‐121‐1030

Associated Nextera Index Kit (24 indices, 96 samples) Illumina FC‐121‐1011

AMPure Beads, 60 mL Beckman Coulter A63881

8M Guanidine HCL Sigma 50937

Water, molecular biology grade general lab supplier

Ethanol, molecular biology grade general lab supplier


PerkinElmer 13

AssemblingtheVersaMoverThe Versa Mover system can move a plate among the three front positions of the NGS Express deck.

This provides automated plate movement among the magnet tile, heating/cooling tile, as well as a

standard tile. The following chart provides information on the components of the Versa Mover system

so you can understand how it works. Use the Place page within the NGS Express software for step‐by‐

step instructions on placement of each piece on the deck of the instrument.

The VersaLift tool has two functions: it links the

For error‐free operation, please:

Place a universal (un‐notched) disposable lid in the lifting tool.

Make sure the plate type you are using matches the Inheco adapter plate type.

Verify that the position of each part matches that shown in deck setup drawing in the software

before starting each run.

Chart:ImagesandDescriptionsofVersaMoverHardware

LabwareDescription

ComponentImage(s) AdditionalInfo

Base‐ this is a

custom support

tile that is

placed on the

deck to hold a

Lifting Tool

w/Lid and/or a

plate in the

carrier Basket

Requires the Basket in order to

hold a plate.

Note the ‘FRONT’ engraved in the

base and make sure it faces the

user.

Base + Basket‐

this pair of

labware holds a

plate on the

Working Tile.


PerkinElmer 14

Base + Magnet‐

this labware

can support the

lifting tool with

lid.

Base + Magnet

+ Basket‐ this

3‐piece

assembly forms

the “Magnet

Tile” and holds

a plate.

VersaLift‐ this

labware is the

‘handle’ for the

basket and also

holds the

disposable lid.

A lid MUST be present for the

Versamover to work correctly.

The spring clip on the front of the

lifting tool allows you to replace

the lid as desired.

The plastic pieces may be putty

colored on your hardware.

Inheco + 96‐V

Adapter Tile‐

special

geometry to

match the

StorPlate 96‐V

from

PerkinElmer.

Photograph of Inheco + inserted 96‐V adapter on

the deck.

Image of 96‐V adapter only


PerkinElmer 15

Pre‐RunInstrumentChecklistThe day before your automation run, the following steps should be performed:

Place the insert for the IsoRack chiller block insert into a ‐20C freezer. It is recommended that

you place the block upside down to keep the bottom foil surface as flat as possible.

Fill the system liquid carboy with de‐ionized water (house‐distilled or de‐ionized; Milli‐Q is also

suitable). This will allow the system liquid to de‐gas overnight, which will reduce air bubbles in

the line during the next day’s run.


PerkinElmer 16

RunningtheNexteraDNASamplePrepMethodonNGSExpress

ExperimentalPlanningBefore starting the automated run, a number of details must be determined, including:

How many samples you will be running?

Which barcoded adapters will you use on which samples?

Will you normalize and/or pool your samples?

InputFilesThe Nextera Option for NGS Express performs some pipetting steps based on user‐provided input files,

which are described below. For efficient sample processing, be ready to supply these files during the

run.

IlluminaSampleSheetThis is a csv file that can be generated using the Illumina Experiment manager. The purpose of this file is

to designate which Index adapters are to be added to each sample. You must have this file ready before

starting the protocol 2 of the Nextera Application. The NGS Express software extracts the indexing

information from the Illumina Sample Sheet‐ 24 Samples.csv file (or another file that you select) to

generate the index pipetting worklist. An example of this sheet is provided in C:\Packard\Janus\Bin and

is shown below.

Figure 9. Illumina Sample Sheet with example index data.


PerkinElmer 17

This sheet can be created by the Illumina Experiment Manager (IEM), or you can edit the provided

template, Illumina Sample Sheet‐24 Samples.csv. It must be a .csv (comma‐separated values) file and

the format should be the same as the “Illumina Sample Sheet” as exported by IEM version 4. Specifically:

First row of data is in row 22

Well ID is in column 4 (D)

Index 1 (I7 info) is in column 5 (E)

Index 2 (I5 info) is in column 7 (G)

Include in the file only those rows/wells that you wish to process

Your file need not be named as the one above, but it must be in CSV format and have the .csv extension.

If you have a different data structure (data locations within the file are different from above) that you

wish to routinely use, the protocol will need to be edited.

Normalization&PoolingWorklistIn order to perform automated library normalization and pooling, a .csv file that tells the NGS Express

the concentrations of your libraries and how they should be pooled must be provided. Library

concentrations (determined by your method of choice) are entered into the worksheet and the pool

number is specified. The NGS Express automatically calculates the required volumes of sample and

diluent to pipet in order to normalize the samples. It is recommended that you edit the provided

template NexteraNormPoolingWorklist.csv and change only the numbers in columns C and F. The

template file is located in C:\Packard\Janus\Bin and is shown below:

Figure 10. Nextera normalization and pooling worklist.


PerkinElmer 18

To edit the template,

Open the file in Microsoft Excel.

Delete any rows in the worksheet that correspond to wells that you will not be using.

Edit the concentration numbers in column C to match your resulting library concentrations (you

will not have this information until doing the analysis after protocol 3).

If you wish to pool the samples, enter the pool number(s) in column F.

Save the file in csv (Microsoft Excel Comma Separated Values File) format. It is not required that

you keep the file name, just the format. You will browse to and select this file when starting the

normalization and pooling step (protocol 4).

ReagentandSamplePreparation

ReagentTubesThere are two types of tubes used for the reagents, 0.5mL and 2.0mL. It is important to use the correct

tube type for each reagent to ensure that the pipet tip reaches each reagent with minimal dead volume.

Reagent Vessel Type and Number

Tagmentation DNA Buffer (TD) 2.0 mL (1)

Tagmentation Enzyme (TDE) 0.5 mL (1)

AMPure XP Beads 2.0 mL (4)

Guanidine HCl, 8M solution 2.0 mL (4)

Resuspension Buffer (RSB) 2.0 mL (1)

Nextera PCR MasterMix (NPM) 0.5 mL (1)

PCR Primer Cocktail (PPC) 0.5 mL (1)

Index 1 primers (N7XX) 0.5 mL (1‐6)

Index 2 primers (N5XX) 0.5 mL (1‐4)

ethanol, 80% solution 150mL trough (1)

DeterminingReagentVolumesConsult the calculator worksheet “Reagent Volume Calculator‐Nextera DNA Sample Prep.xlsx” for

required reagent volumes, or use the excel worksheet to calculate the volumes required for the number

of libraries you will be preparing. To use the calculator, simply enter the number of Samples you wish to

run in cell A2 (yellow highlighted number in Figure 11), and the calculator will determine the correct

volume of each required reagent and display them in column G. Please note that for the AMPure beads

and Guanidine reagent, four tubes at the given volume must be prepared.


PerkinElmer 19

Figure 11. Reagent volume calculation worksheet. Upon entering the number of samples

to be run in the yellow box, the volumes of all reagents are calculated. Note the

total per‐day reagent volumes for beads, RSB, and ethanol at the bottom of the chart.

For each index adapter, the volume should be calculated as follows:

((Number of libraries using the index) x 5 µL) + 3 µL

For example, if you are preparing four libraries and you are using I701 as Index 1 for all four and I501‐

I504 for Index 2, the amount of I701 you would transfer to the tube would be (4 x 5) + 3 = 23 µL and for

each of the I5xx adapters you would need (1 x 5) + 3 = 8 µL.


PerkinElmer 20

NGSReagentRackSome reagent tubes will be placed in the NGS Reagent Rack which is mounted on the left side of the

deck.

Index adapters in 0.5 mL tubes

Resuspension Buffer (RSB) in a 2.0 mL tube

The scalloped depressions at the outer edges hold the reagent tube caps when the tube is on the deck.

Tubes are not included in the JAA image (Figure 12), so consult the Reagent Rack Maps for locations of

tubes at each step in the method.

Make 80% ethanol fresh using molecular biology‐grade reagents and place it in the trough. Keep the cap

on the trough until you are ready to start the run.

Figure 12. Left‐mounted labware: reagent trough and microtube rack.

IsoRack24‐WellCoolingBlockOther tubes will be placed in the IsoRack cooling block Figure 13 which is then placed on a support tile

on the deck. For correct tube placement always use the cooling insert. When properly assembled, the

chiller insert will be sitting about 1cm above the deck. Keep the block insert in the freezer when not in

use.

Some reagent tubes will be placed in the IsoRack cooling block, including:

Enzymes in 0.5 mL tubes

Guanidine HCl in 2.0 mL tubes

AMPure reagent (SPRI beads) in 2.0 mL tubes.


PerkinElmer 21

Tubes are not shown in the JAA image, so consult the Reagent Rack Maps for locations of tubes at each

step in the method.

Figure 13. Rendering of the assembled IsoRack + IsoPack cooling block.

SamplesPlace your samples in a StorPlate‐96 V‐bottom plate. Each sample should contain 40‐50ng of DNA in a

total volume of exactly 20 µL. Samples must be placed into the plate in order of columns, as shown

below:

Figure 14. Sample locations at the start of the Nextera 1 protocol.

Samples must be arranged top to bottom starting in well A1.


PerkinElmer 22

InstrumentPreparationClean, degassed water must be available throughout the instrument operation to maintain proper

pipetting performance and avoid delays in the protocol. The following actions must be performed each

day, prior to starting any protocol runs.

CheckFluidLevels Check the System Liquid level‐ Check the volume of water in the carboy. If it is less than half full,

you should add fresh deionized water to it.

Check the waste container‐ to avoid overflow, make sure the waster container is empty or near

empty.

PrimetheSystemAll air bubbles must be removed from the fluid path for optimal pipetting performance. To flush the

system liquid path,

1) Open the NGS Express software by clicking on the icon.

2) Go to the Maintain tab and find the “Prime the Varispan Fluid Path” activity.

3) Click the Start Maintenance button to initiate the system priming routine.

Figure 15. Maintain tab shown with Prime Varispan path highlighted.


PerkinElmer 23

RunningtheAutomatedNexteraProtocolsNow that your experiment is planned and your reagents and system are ready, you can proceed with the

automated run.

Protocol1‐TagmentationandClean‐UpThis protocol performs the following actions on your samples:

Addition of tagmentation buffer and enzyme, and mixing by pipetting the samples several times.

• On‐deck incubation of the tagmentation reactions. To eliminate a manual interaction, the

tagmentation takes place on the deck. Both time and temperature are increased relative to the

manual method. The results are equivalent.

• Double‐sided SPRI cleanup of the tagmented samples. Chilled Guanidine HCl is added to

dissociate the tagmentase enzyme from the DNA and cool the reaction. With guanidine as a

denaturant, a double‐sided SPRI can be used to clean up the tagmentation reaction and avoid

the manual spin‐column method.

Consult the Workflow Chart for more details on the steps being performed by the NGS Express.

To Start the Protocol,

1. Open the NGS Express software by clicking on the icon. The list of available

protocols will appear.

2. Select the protocol “Nextera 1_Tagmentation and Clean‐Up” by clicking on it

once.

3. Questions related to this protocol will appear in the lower panel.

a. Enter the number of samples you want to process.

b. If you wish to generate a Run Report, leave the checkbox checked. If checked, a report

will be generated including the following:

i. Name or User ID‐ this optional file allows you to record which operator is

running the instrument.

ii. Kit Barcode‐ use the handheld barcode scanner or type in the kit barcode. This

information will be saved in the Run Report.


PerkinElmer 24

4. Place the labware and reagents on the deck.

a. Click on the Place icon in the top green bar.

b. Follow the instructions in the Place tab of the software for placing support tiles, tips,

and plates on the deck. Note that your sample plate must be in the basket in the front

left position on the deck, as shown in Figure 17.

Figure 17. Place page of NGS Express software showing the location of the sample plate on the deck for Nextera protocol 1.

Figure 16. Select Protocol Screen, with Nextera 1 protocol selected and related questions

shown.


PerkinElmer 25

c. Place the tube of Resuspension buffer (RSB) in the lower right position of the left‐

mounted reagent rack. See Figure 18.

d. Place the prepared reagent tubes in the IsoRack chilling block according to the diagram

in Figure 18.

5. Run the Protocol

a. Click on the Run icon in the top green bar.

b. Click the Start button.

c. You will be asked to verify/change tip counts. If you are placing full tip boxes on the

deck, make sure that each box shows 96/0 tips in the software. If you have full tip boxes

on the deck but not in the software, click the Fill button for each tip box that is full on

the deck.

Figure 18. Diagrams of reagent placement for Protocol 1.


PerkinElmer 26

Figure 19. Interface for refilling tip boxes in JAA.

d. Click OK to commence the run.

e. Verify the location of the samples, and then click OK.

Consult the Workflow Chart for details on the steps being performed by the NGS Express.

When the protocol is complete, a message will appear:

Figure 20. NGS Express screen showing completion of protocol 1.


PerkinElmer 27

Your samples will be in the plate on the magnet tile, in the wells shown below:

Figure 21. Sample locations at the completion of protocol 1.

It is not necessary to remove the sample plate‐ the next protocol will transfer the samples from the

plate on the magnet to the PCR vessel.


PerkinElmer 28

Protocol2‐PCRSetupwithDualIndexingThis protocol performs all of the pipetting required to set up the PCR reactions in a PCR plate or strip

tubes, including:

Addition of Nextera PCR Master Mix to PCR plate/tubes

Addition of PCR Primer Cocktail

Addition of I7 and I5 index adapters

Transfer of tagmented, cleaned‐up DNA to the PCR tubes/plate

This protocol requires some user inputs: the choice of plate or strip tubes for the PCR vessel, and

selection of index adapters to be used for each sample. For the latter, the Illumina Sample Sheet is

used to provide the indexing information. A description of this file is located in the Illumina Sample

Sheet section on page 16 of this document.


1. Verify that the correct Illumina sample sheet is on the NGS Express computer.

2. Click the Select icon in the green bar near the top of the screen.

3. Select the protocol “Nextera 2_PCR Setup with Dual Indexing” by clicking on it once.

4. Questions related to this protocol will appear in the lower panel.


PerkinElmer 29

a. Browse to the Illumina Sample Sheet. The default location is in C:/Packard/Janus/Bin

but you may browse to the location of the current file.

b. Select the destination labware. You may choose a Hard‐shell PCR plate or PCR tubes in a

hard‐shell plate. Preparing the samples directly in PCR tubes is useful if you have only a

few samples or your thermocycler does not accommodate full‐skirt plates.

c. If you wish to generate a Run Report, leave the checkbox checked. If checked, a report

will be generated including the following:

i. Name or User ID‐ this optional file allows you to record which operator is

running the instrument.

ii. Kit Barcodes‐ use the handheld barcode scanner or type in the barcode(s) on the

boxes of the Nextera Sample Prep Kit as well as the Indexing Kit. This

information will be saved in the Run Report.


PerkinElmer 30

iii. A copy of the selected Illumina Sample sheet will be archived with the Run

Report files.

5. Place Labware and Reagents

a. Click on the Place icon in the upper green bar and follow the instructions to place the

labware on the deck.

b. Note the replacement of the 175 µL tips in position B4 with 25 µL tips.

c. The samples from step 1 should still be on the magnet tile; this protocol will transfer the

DNA from columns 7‐9 of the plate on the magnet tile to the PCR vessel chosen above.

Figure 22. Place screen showing placement of source samples for the start of Nextera protocol 2.

d. Place the prepared reagent tubes in the left‐mounted custom rack and IsoRack

according to the diagram in Figure 23.


PerkinElmer 31

e. Place the correct labware (a hard‐shell PCR plate with or without strip tubes) on the

center rear support tile at location B4. Please note:

i. A hard‐shell PCR plate must be present regardless of whether you are using

tubes or preparing reactions directly in the plate.

ii. If tubes are used, they must be placed in the wells of a hard‐shell plate in order

of columns as shown in the well map below.

Figure 24. PCR setup destination well locations. If using PCR tubes they must

be placed in the wells of a hard‐shell PCR plate in the positions shown.

6. Run the Protocol



c. You will be asked to verify/change tip counts. This protocol requires five 25 µL tips per

sample. If you are running 8 samples or less you do not need to replenish any tips.

Figure 23. Reagent tube placement for Nextera protocol 2.


PerkinElmer 32

d. You will be asked to verify sample and plate locations. Click OK to proceed.

AmplifytheSamplesWhen the PCR setup protocol is complete, you will see a message as shown below.

To Start the Amplification,

1) Carefully remove the PCR tubes/plate from the center rear deck position.

2) Seal the plate with thermocycler‐compatible film. If using tubes, cap the tubes. Make sure to

maintain the orientation of the strip tubes.

3) Run the amplification protocol as specified in Illumina’s Nextera Sample Preparation Guide.


PerkinElmer 33

Protocol3‐Post‐PCRClean‐UpAfter amplification is complete, the NGS Express will perform a single SPRI clean‐up of the 50 µL PCR

reaction. Samples are manually transferred to a StorPlate, then 35 µL AMPure reagent (a 0.7x ratio) is

added by the automated system. The SPRI ratio differs from the manual protocol but provides the

appropriate size range of fragments in the final libraries.

To Prepare the Samples for the Next Step,

1. Remove the plate/tubes from the thermocycler.

2. Centrifuge the plate briefly to collect all of the liquid in the bottom of the wells.

3. Transfer the samples to a clean StorPlate 96‐V bottom plate, using the well locations shown

below.

Figure 25. Well diagram showing locations for placing amplified libraries to start Nextera protocol 3.


1. Click the Select icon in the green bar near the top of the screen on the NGS Express.


PerkinElmer 34

a. Select the protocol “Nextera 3_Post‐PCR CleanUp” by clicking on it once. Questions

related to this protocol will appear in the lower panel.

b. Provide the number of samples, whether you wish to have the system mix the beads

prior to addition to the samples, and whether you’d like to generate a Run Report.




b. Note that the Versa Tool Basket may still be on the magnet‐ if so, you must swap the

positions of the Versa Lift (lid) and the basket so that the Basket and your sample plate

are on the Working Tile (front left position).


PerkinElmer 35

c. If you are processing 12 or fewer samples, you can rotate the waste plate 180° and re‐

use it to collect the waste for this SPRI clean‐up.

d. Place the prepared reagent tubes in the left‐mounted custom rack and IsoRack

according to the diagram in Figure 26.

Figure 26. Reagent placement for Nextera protocol 3.


PerkinElmer 36

3. Run the protocol



c. You will be asked to verify/change tip counts. Note that the tips counts from the 175 µL

tips will be remembered from step 1, so you can start with partially full tip boxes if you

are running fewer than 12 samples.

d. Click OK to continue.

Upon completion you will see a message saying that the samples are ready. The samples (30 µL each)

are located in columns 4‐6 of the plate, as shown in Figure 27 below:

Figure 27. Diagram of purified library locations after completion of Nextera protocol 3.

AnalyzeYieldandQualityoftheLibrariesAt this point the libraries are ready for quantitation and sizing analysis. Use your preferred method for

library quantification (Pico Green or Qubit) and size traces (LabChip GX with DNA Nigh Sensitivity chip,

or equivalent). Yields can vary from 3ng/µL up to 20ng/µL.

Convert the concentration data to nanomolar (nM) using the method stated in the “Validate Library”

section of Illumina’s Nextera DNA Sample Preparation Guide.


PerkinElmer 37

Protocol4‐NormalizationandPoolingNGS Express performs normalization of samples based on the concentration data, read from a csv file

that you will provide when starting the protocol. In addition it is possible to pool the samples.

Normalization and pooling are performed separately; the samples are first normalized to 4nM, and then

equal volumes of the normalized samples are combined to create the pools. You will choose at run time

whether you wish to normalize and/or pool the samples.

Before running the protocol, you must first edit the NexteraNormPoolingWorklist.csv file with your

actual concentration data in units of nM. It is recommended that you edit the provided template and

change only the numbers in columns C and F. The template file is located in C:\Packard\Janus\Bin and is

shown below:

To edit the template,

Open the file in Microsoft Excel.

Delete any rows in the worksheet that correspond to wells that you will not be using.

Edit the concentration numbers in column C to match your resulting library concentrations in

nanomolar (nM).

o IMPORTANT‐ the concentrations must be the actual nanomolar concentration in each

well and should not contain any units.

If you wish to pool the samples, enter the pool number(s) in column F. The pool number

corresponds to the well number in the Pooled plate according to the well map below:


PerkinElmer 38

Save the file in csv (Microsoft Excel Comma Separated Values File) format. It is not required that you

keep the file name, just the format. You will browse to and select this file when starting the protocol.

NOTE: This protocol is pre‐configured to prepare 25 µL of DNA normalized to 4nM, per Illumina’s

Nextera protocol. To change these settings, consult the DNA Normalization User’s Guide or contact your

product specialist.


1. Click the Select icon in the green bar near the top of the screen on the NGS Express.

Select the protocol “Nextera 4_Normalization and Pooling” by clicking on it once. Questions related to

this protocol will appear in the lower portion of the screen, as shown below.

a. You have the option to normalize the samples, pool the samples, or perform both

procedures. If you choose to normalize and pool the samples, the normalized samples

will be used to create the pools.


PerkinElmer 39




b. The DNA source plate (which is the product of protocol 3) must be placed on the center

back tile of the deck, as shown in Figure 28.

c. Two clean PCR plates must be placed on the deck as well to hold the normalized and

pooled samples. The normalized samples will be prepared in the plate in the center

deck position and samples will be pooled into the plate on the basket (left front

position).

d. The dilution buffer (molecular biology‐grade water, TE, or RSB) must be placed in a 2.0‐

mL tube in position 18 of the left‐mount rack (see Figure 26).

Figure 28. Place view for protocol 4 showing the position of the source DNA plate.

3. Run the protocol



c. You will be asked to verify/change tip counts. You will need a maximum of 4 (four) 175‐

µL tips and 48 25‐µL tips so it may not be necessary to refill the boxes. Do not click the

‘fill’ button unless you have actually placed a full tip box on that deck position.

d. Click OK to continue.

Upon completion of the protocol, the normalized samples will be in the plate in the center deck position

(highlighted in Figure 29) and the pooled samples will be in the plate in the Versa Mover basket (left


PerkinElmer 40

front position).

Figure 29. Deck layout for normalization and pooling. Normalized samples are in the center PCR plate (highlighted), source

samples are in the center back StorPlate, and pooled samples (if prepared) will be in the PCR plate in the basket (front left)

position.


PerkinElmer 41

AdvancedUsersGuideThe NGS Express has been designed to run wholly within the JANUS Application Assistant (JAA)

interface. In most cases customers will not need to enter the protocol development software

(WinPrep). However the following information will be helpful if you find you need to make some

changes to the protocols you are running.

NOTE: All files are installed as read‐only to prevent accidental changes. It is recommended that if you

wish to change a protocol or labware definition, you back up the original file(s), and then make the files

of interest read‐write via the Properties function in Windows.

CustomLabware

Left‐mountreagentrackThe NGS Custom Rack has 18 tube positions plus a trough position. For

Nextera, each well is configured for a specific tube type:

Wells 1‐16: Sarstedt 0.5 mL tube

Wells 17‐18: Sarstedt 2.0 mL tube

Well 19: 150 mL trough

If not using Sarstedt tubes, you may need to edit the well bottom of the

well(s) affected. Note the well numbering, shown in Figure 30, to make

certain that you are editing the correct well.

The left‐mounted hardware is not physically attached to the calibrated

deck; in order to optimize pipetting positions, the labware definition file

“NGS Reagent Rack Custom_Nextera DNA Sample Prep (left edge).lab”

may need to be edited.

NOTE: to maintain efficient pipetting, it is vital that the X position be

identical across all wells in column 1, as well as column 2; in addition, the Y

spacing among wells 1‐16 must be 18 mm.

IsoRackNote that the well numbering in the labware maintains the WinPREP

convention and does not reflect the numbering imprinted on the rack

itself. Figure 31 shows the well numbering and tube type for this piece of

labware, as determined by the labware file "IsoRack 24 Custom_Nextera

DNA Sample Prep.lab".

Figure 30. Custom rack

well numbering.


PerkinElmer 42

Figure 31. Well numbering and tube capacity (mL) for the

labware file "IsoRack 24 Custom_Nextera DNA Sample Prep.lab"

DeckPlacementofLabwareNote that the left‐mounted labware overlaps Column 1 in the WinPREP software. In order to place

labware items in column 1 on the deck, hold down the Alt key while dragging with the mouse to allow

placement of multiple pieces of labware on the same spot or overlapping to any degree.

VersaMoverInformationIf you open any of the NGS protocols in WinPREP you will notice multiple layers of labware where the

Versa Mover parts are (Row F). In order for the Versa Mover to work correctly multiple labware, each

with its own support labware, must be present in those positions. If you need to change the labware

(for example replacing a plate type), be very careful to arrange all of the other labware correctly as it

was in the original protocol. Additional information about Versa Mover functionality and labware can

be found in the Installation and Support Guide (Document No. 8860049).

ProtocolLogicElementsThis section describes unique scripts and protocol logic built into the Nextera protocols.

Nextera2_PCRSetupwithDualIndexingThe dual‐indexing capability in the Nextera 2 protocol is driven by a MSL script function that generates

two worklists (one for each index) based on the Illumina Sample Sheet. In the WinPREP protocol, the

Generate Pipetting Worklist node controls how this user input file is read and converted into the two

worklists that drive the addition of the indexing primers. As shown below, the worklist can be

configured for different input file formats, a different source rack, etc.


PerkinElmer 43

Notes on the script arguments:

The argument szMasterWorklistPath is asking for the name of the input file. This is set to Var:

Ask User so that you can browse to a unique file each time. The file Illumina Sample Sheet‐ 24

samples.csv is provided as a demonstration file as well as a template illustrating the correct

format and structure.

szReagentTubeNumbersPath.csv is the argument calling for the location of the reagents by tube

number. It does not specify which rack, only the position within the reagent rack. The value,

NexteraRackSetup.csv ,states the location of each individual index tube in a rack. This file can

be edited if required to use a different set of barcodes. There is no header for this file, as shown

in Figure 32.

The argument szPipettingWorklistPath is where the name of the output file is specified. Note

that for the value given, two files will be generated named with 1 or 2 at the end. For example,

a value of ReagentPipettingWorklist.csv will yield, upon protocol execution, two files:

ReagentPipettingWorklist1.csv and ReagentPipettingWorklist2.csv. Two separate worklists are

generated so that each destination well is visited only once per procedure.

The argument szDestinationPlate is asking for the deck name of the plate where the PCR

reactions will be set up. The value Var: Ask User in this case allows the customer to choose

whether to prepare the reactions in a PCR plate or tubes sitting in the plate. The initial user

query entries match the names of the deck labware and this information is put into the reagent

pipetting worklists.

The argument szReagentRack refers to the deck name of the labware where the indexes are

located. By default this is the NGS Custom Rack, which is the left‐mounted reagent rack. There

are sixteen (16) positions for 500 µL tubes in this rack that are available for indexes. If you wish


PerkinElmer 44

to use more indexes you will need to designate a different source rack by changing the value for

this argument.

The argument nColumnOffset refers to the destination wells. By default (0) the PCR reactions

will be set up in the first 3 columns. If you want to set up the reactions in another location on

the plate, you would change the value to an integer representing the number of columns to

move the samples to the right. For example if nColumnOffset = 6, the PCR reactions will be set

up starting in Column 7.

The last four arguments describe the structure of the input data (Master Worklist) file. The

defaults correspond to the Illumina Sample Sheet as exported by Illumina Experiment Manager

File Version 4 (software version 1.3).

Figure 32. NexteraRackSetup.csv

Upon running the protocol, all of the rack, plate, and reagent information is synthesized, the reagent

pipetting worklists get generated, and pipetting of indexes is executed accordingly. A view of one of


PerkinElmer 45

the pipetting worklists is shown below.

Figure 33. Example of reagent pipetting worklist generated in protocol 2, showing the choice of destination plate and well

location for each index adapter to be pipetted.

Nextera4_NormalizationandPoolingThe normalization and pooling protocol utilizes the “DNA Normalization for EnSpire” option, which is

included in the NGS Express.

1. The sample information must be provided in the form of a worklist file. The file must be in csv

format with data located as described in the Normalization & Pooling Worklist section on page

17. If you wish to change the default file structure (such as location of data within the file) you

can edit the “Create Normalization Worklist” node of the protocol in WinPREP.


PerkinElmer 46

The protocol “Nextera 4_Normalization and Pooling” has been configured for Nextera libraries, as

shown in Figure 34 below.

Figure 34. Normalization parameter settings as shown in WinPREP.

If you need to change any of these parameters, you will need to edit the protocol in WinPREP. The

dialog box is found in the WinPREP protocol under step 1, “Set Normalization Parameters”. Open this

procedure and click Edit Parameters to open the dialog box shown in Figure 34.

For additional details on the normalization function, see document no. 8860023B, “JANUS Nucleic Acid

Normalization for EnSpire” which is available on the normalization installer CD (P/N 8701686/B).


PerkinElmer 47

Appendix

Labware&ConsumablesOrderingInformation

TipsDisposable Tip‐ 25ul, Clear, Sterile Filter Tips, qty 960 PerkinElmer 6000689

Disposable Tip‐ 175ul, Clear, Sterile Filter Tips, qty 960 PerkinElmer 6000685

PlatesandLidsStorPlate, PP, V‐bottom, 450 µL capacity, pack of 50 PerkinElmer 6008290

Hard‐Shell PCR plates, 96‐well, pack of 50 Bio‐Rad HSP‐9631

Lid‐ Universal, polystyrene, pack of 100 Seahorse (various distributors) 200856‐100

Tubes,Caps,andRacksScrew‐cap microtube, 2.0 mL capacity Sarstedt 72.609.711

Screw cap microtube, 0.5 mL capacity Sarstedt 72.730.711

Caps for microtubes, blue Sarstedt 65.716.723

Cooling Block, 24 positions, for 1.5 mL tubes Eppendorf 022510240


PerkinElmer 48

ListofFilesInstalled/ModifiedbytheNexteraOptionInstallerNew files have been added to the Bin, Labware Files, and Performance Files folders.

Option‐SpecificProtocolFilesAdded: Nextera 1_Tagmentation and SPRI

CleanUp.MPT

Nextera 2_PCR Setup with Dual Indexing.MPT

Nextera 3_Post‐PCR CleanUp.MPT

Nextera 4_Normalization and Pooling.MPT

Illumina Sample Sheet‐ 24 Samples.csv

NexteraNormPoolingWorklist.csv

NexteraRackSetup.csv ReportConfig_Nextera1.csv ReportConfig_Nextera2.csv ReportConfig_Nextera3.csv ReportConfig_Nextera4.csv

CommonFilesAdded: AlignTips_NGS.MPT

DailyClean_NGS.MPT

FlushSysLiq_NGS.MPT

JAA AlignTips_NGS.MPT

JAA Clean_NGS.MPT

JAA Prime_NGS.MPT

PickupTipTest_NGS.MPT

Random XY Test_NGS.MPT

Left Rack Test.MPT

Versa Mover Library.MPT

Versa Mover Test.mpt

NGS.s NGSReporting.s NGS Express_Normalization.MPT

NGS Express_SPRI PCR CleanUp.MPT

NGS Express_SPRI Size Selection.MPT

NGSExpress_NormalizationWorklist.csv

ReportConfig_NGS_Normalization.csv

ReportConfig_NGS_PCR_Cleanup.csv ReportConfig_NGS_SizeSelection.csv NGS_PCR 96_WinPREP_Template.MPT

NGS_StorPlate 96‐V_WinPREP_Template.MPT

NGSReportingTemplate.MPT

LabwareFiles‐Consumables: IsoRack 24 Custom_Nextera DNA Sample

Prep.lab

NGS Reagent Rack Custom_Nextera DNA

Sample Prep (left edge).lab

Bio‐Rad Hard‐Shell 96 PCR for Magnet.lab

Bio‐Rad Hard‐Shell 96 PCR Skirted (blue).lab

Invitrogen MicroAmp 96‐PCR with base.lab

IsoRack 24‐well Cooling Block with 1500uL tube.lab



IsoRack 24‐Well Custom Cooling Block.lab

PCR tubes in Bio‐Rad Hard‐Shell Plate.lab PerkinElmer StorPlate 96‐V (PP).lab

Seahorse 10mL Square 24‐Pyr (PP).lab

175ul Conductive Filter RoboRack Tips.lab 175ul Non Conductive Filter RoboRack Tips.lab

25ul Conductive Filter RoboRack Tips.lab 25ul Non Conductive Filter RoboRack Tips.lab

LabwareFiles‐CustomHardware: Inheco 96‐PCR Plate Support 33mm.lab

Inheco 96‐V Plate Support.lab Rack_18 Custom Microtube+Trough (left

edge).lab

VM_Base_LiftSupport.lab

VM_Base_PlateSupport.lab

VM_Inheco96PCR_LiftSupport.lab

VM_Inheco96PCR_PlateSupport.lab

VM_Inheco96V_LiftSupport.lab

VM_Inheco96V_PlateSupport.lab

VM_Mag_LiftSupport.lab

VM_Mag_PlateSupport.lab

VM_VersaLift.lab


PerkinElmer 49

Washbowl+Tip Chute (left edge).lab

Tip alignment plate.lab

PlateMoverFiles‐DoNotEdit: HeaterToMagnet.csv

HeaterToReaction.csv HeaterToReactionLidatHeater.csv LidFromMagnetToHeater.csv

LidFromMagnetToHeaterDropLid.csv

LiftZ20.csv LiftZ24.csv LowerZ24.csv

MagnetToWorking.csv

ParkLidHeaterToMagnet.csv

ParkLidHeaterToWorking.csv

ParkLidMagnetToWorking.csv

ParkLidWorkingToMagnet.csv

PlateMagnetToHeaterLidatR.csv

ReactionToHeater.csv ReactionToHeaterLidOn.csv ReactionToMagnet.csv

PerformanceFilesThe following performance files have been created expressly for the NGS Express and were validated

with 250‐µL syringes.

AMPure XP_Blowout_DT‐175uL_S250.prf

AMPure XP_Blowout_DT‐25uL_S250.prf

AMPure XP_Waste_DT‐175uL_S250.prf

Ethanol 80%_Blowout_DT‐175uL_S250.prf Ethanol 80%_Waste_DT‐900uL_S250.prf

Glycerol 10%_Blowout_DT‐25uL_S250.prf Glycerol 10%_Waste_DT‐25uL_S250.prf

PEG 20%_Blowout_DT‐25uL_S250.prf PEG 20%_Waste_DT‐175uL_S250.prf

PEG 20%_Waste_DT‐900uL_S250.prf

Water_Blowout_DT‐175uL_S250.prf

Water_Blowout_FT_S250.prf

Water_Waste_DT‐175uL_S250.prf

Water_Waste_DT‐900uL_S250.prf

Water_Waste_FT_S250.prf

ModifiedWinPREPFilesThe following common WinPREP files (in Bin unless otherwise noted) are modified by the installer, and

are backed up prior to modification:

Categories.xml

CleanupActivityList.xml

DiagnosticTests.csv LabwareImagesInfo.XML

MaintainActivityList.xml

Msl.ini

MSLEXT.csv

C:\Packard\JANUS\InstrumentLayout\InstrumentLayout.xml

Nextera DNA Library Preparation for NGS Express · Application Guide- Nextera DNA Library...

Documents

Transcript of Nextera DNA Library Preparation for NGS Express · Application Guide- Nextera DNA Library...