Download - KAPA qPCR Library Quantification for NGS Express · Application Guide- KAPA qPCR Library Quantification for NGS Express PerkinElmer 8 KAPA qPCR MMX (KAPA qPCR Master Mix + 10X Primer

KAPA qPCR Library Quantification

for NGS Express

Application Guide

PerkinElmer 2200 Warrenville Road

Downers Grove, IL 60515

[email protected]

Document Number 8860051

BETA

Copyright ©2012, PerkinElmer, Inc.

All Rights Reserved

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.

SYBR® and MicroAmp® are registered trademarks of Life Technologies Corporation.

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

Application Guide- KAPA qPCR Library Quantification for NGS Express

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TableofContents

Required Materials List ............................................................................................................................. 4

NGS Express Kits & Reagents ................................................................................................................ 4

Labware & Consumables ...................................................................................................................... 5

NGS Express Custom Deck Tiles and Racks ........................................................................................... 6

Reagent and Sample Preparation ............................................................................................................. 7

NGS Express Well Maps ............................................................................................................................ 8

KAPA qPCR Sample Plate Well Map ...................................................................................................... 9

KAPA qPCR Reagent Rack Well Map ................................................................................................... 10

KAPA qPCR Dilution Plate Well Map ................................................................................................... 11

KAPA qPCR Assay Plate Well Map ....................................................................................................... 12

Advanced Protocol Logic: Validation of User Entries ............................................................................. 13

Error Checking: Required Entries ........................................................................................................ 13

Error Checking: Sample Count ............................................................................................................ 16

Error Checking: Skipped Samples ........................................................................................................ 17

Error Checking: Duplicate Samples ..................................................................................................... 18


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RequiredMaterialsList

Materials Required but Not Supplied:

RequiredKits&ReagentsItem (Kits & Reagents) Vendor/Part Number

KAPA SYBR® FAST Universal qPCR Kit

1 x 1ml Illumina Primer Premix (10X)

6 x 80ul Illumina DNA Standards

KAPA Biosystems

KK4824

(KK4601+ KK4808)

KAPA SYBR® FAST ABI Prism qPCR Kit



KAPA Biosystems

KK4835

(KK4606 + KK4808)

KAPA SYBR® FAST Bio‐Rad iCycler qPCR Kit



KAPA Biosystems

KK4844

(KK4607 + KK4808)

KAPA SYBR® FAST LightCycler qPCR Kit



KAPA Biosystems

KK4854

(KK4610 + KK4808)

Water, molecular biology grade

(Used for qPCR Water Controls)

general lab supplier

10mM Tris‐HCL, pH 8.0 +0.05% Tween 20

(Used for Serial Dilution Buffer)

general lab supplier

MicroAmp® Optical Adhesive Film (1 Film) Life Technologies

4311971


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Labware&ConsumablesThese materials are included in the NGS Express system package.

Item (Labware & Consumables) Vendor/Part Number

Disposable Tip‐25ul, Clear, Sterile Filter

Tips, (4 Boxes)

PerkinElmer

6000689

Hard‐Shell PCR Plates, 96‐well, (2 Plates) Bio‐Rad

HSP‐9631

MicroAmp Optical 96‐Well Reaction Plate (1

Plate)

Life Technologies

N801‐0560

MicroAmp Splash Free 96‐Well Base (1

Support)

Life Technologies

4312063

Lid‐ Universal, Polystyrene, (1 Lid) Seahorse (various

distributors)

200856‐100

Screw‐Cap Microtubes, 2.0mL capacity (16

Tubes)

Sarstedt

72.609.711

Caps for Microtubes, blue (Optional for

Reagent Storage)

Sarstedt

65.716.723


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CustomDeckTilesandRacksThese materials are included in the NGS Express system package.

Item (NGS Supports & Deck Tiles) Vendor/Part Number

NGS Reagent Rack (1 Rack) PerkinElmer

VersaLift Tool PerkinElmer

96‐Torroid Magnet PerkinElmer

VersaMover Base PerkinElmer

VersaMover Basket PerkinElmer

VersaMover Base PerkinElmer

Inheco 96‐PCR Plate Support 33mm (1

Support; Not used in KAPA Protocol)

PerkinElmer


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ReagentandSamplePreparation

The NGS Express reagent rack requires both kit and non‐kit regents for KAPA qPCR Library

Quantification. A choice of 4 KAPA SYBR FAST qPCR kits is available (Universal, ABI Prism, Bio‐Rad

iCycler, and LightCycler), and include Primer Premix, Illumina DNA Standards, ROX High and ROX Low

dyes. Only 1 kit is required for operation on the NGS Express. Individual kit components can be

purchased/assembled as needed. NGS Express requires the following Setup Guidelines:

Notes: All reagents placed on the NGS Express should be transferred into 2ml Sarstedt screw cap

microtubes. Critical dimensions, including diameters, heights, depths, well bottoms, etc. are pre‐

programmed into the automated workflow; using alternative microtubes could impact assay results and

may cause damage to the NGS Express system. Alternative tube types can be accommodated, but

requires modification to the labware definitions prior to use (consult with your product specialist).

All reagents should be thoroughly thawed and vortexed prior to aliquoting into Sarstedt tubes. Failure

to do so could impact concentrations and subsequent qPCR results.

NGSExpress_DNALibrarySamples:DNA Libraries 1‐8 (10µl): DNA Libraries/Source Samples should be aliquoted into a 96‐WellHard‐Shell

PCR plate. The NGS Express will access samples from these wells and needed to complete the requested

serial dilutions.

1‐8 DNA Libraries (DNA Libraries; Column 1, wells 1‐8 @ 10µl)

NGSExpress_KAPAqPCRReagents:DNA Standards 1‐6 (30µl): Illumina DNA Standards 1‐6 arrive in 80µl aliquots as part of the KAPA SYBR

FAST qPCR kit. These standards will be used undiluted (neat) in the NGS Express protocol.

6 X DNA Standards (DNA Standard; 6 tubes @ 30µl)

H20/Controls (30µl): Water and/or Control reagents are not supplied in the KAPA SYBR FAST qPCR Kit,

and must be supplied independently. Two positions are allocated on the NGS Express Reagent Rack for

these controls. Water blanks and or controls are recommended for baseline (blank) reads during qPCR

analysis; these samples will be dispensed in similar volumes as DNA Standards 1‐6.

Recommended Molecular Biology Grade Water (H20; 2 tubes @ 30µl)

Optional Configuration: Replace one water control with an internal/custom laboratory‐specific

control sample (H20; 1 tube @ 30µl, Control; 1 tube @ 30µl)


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KAPA qPCR MMX (KAPA qPCR Master Mix + 10X Primer Premix); (250µl): KAPA qPCR Master Mix is

composed of sub‐components from the KAPA SYBR FAST reagent kit. These components include a 1ml

tube Illumina 10X Primer Premix, and 5ml bottle of qPCR Master Mix. These components must be

premixed prior to using on the NGS Express.

Notes: The first time you use the KAPA SYBR FAST qPCR Kit, premix the following components. Mix by

vortexing for 10 seconds and record the date and component details.

KAPA SYBR FAST qPCR Kit Component Volume

Illumina Primer Premix (10X) 1ml

KAPA SYBR FAST qPCR MASTER Mix (2X) 5ml

6ml (Total Volume)

KAPA qPCR MMX (KAPA qPCR MMX; 4 tubes @ 250µl)

Dilution Buffer (250µl): Dilution Buffer reagents are not supplied in the KAPA SYBR FAST qPCR Kit, and

must be supplied independently. Four positions are allocated on the NGS Express Reagent Rack for DNA

dilution buffer. Dilution buffer aliquots can be alternatively composed of high‐grade molecular biology

water or an optional Tris‐HCL Tween buffer (Recommended for reducing surface adherence of DNA to

plastic surfaces).

Molecular Biology Grade Water (H20; 2 tubes @ 250µl)

Optional Dilution Reagent: 10mM Tris‐HCL, pH 8.0 +0.05% Tween 20 (Tris‐HCL Tween; 4 tubes @

250µl)

NGSExpressWellMapsThe following pages show the placement of reagents and samples to start the KAPA qPCR run.


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KAPAqPCRSamplePlateWellMap DNA Samples Loaded in Column 1 of DNA Sample Plate

Sample Plate positioned on NGS Express Deck Position [F1]

Samples must be in Bio‐Rad Hard‐Shell 96 PCR Plate


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KAPAqPCRReagentRackWellMap Thaw, Vortex, and Spin Reagents Tubes

Reagents for 1‐8 DNA Libraries, , Standards, Controls w/Triplicate Serial

Dilutions (Total 96 qPCR Reactions)

Reagents must be in 2ml Sarstedt screw cap microtubes

DNA Standard_1 (30µl)






KAPA qPCR MMX (250µl)

Dilution Buffer (250µl)

H20/Control (30µl)


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KAPAqPCRDilutionPlateWellMap Dilution Plate must be EMPTY at start of run

Dilution Plate positioned on NGS Express Deck Position [D1]

Dilutions will be performed in a Bio‐Rad Hard‐Shell 96 PCR Plate

Dilutions selected in the

drop menu will determine

how which dilutions are

used for qPCR setup


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KAPAqPCRAssayPlateWellMap qPCR Plate must be EMPTY at start of run

qPCR Plate positioned on NGS Express Deck Position [B1]

qPCR reactions will be aliquoted in a MicroAmp Optical 96‐Well Plate

Dilutions selected in the

drop menu will determine

the final qPCR Plate format.


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AdvancedProtocolLogic:ValidationofUserEntries

The NGS Express Software will require valid user entries within the NGS “Select” tab questions.

Required entries apply to User Data, KAPA SYBR FAST qPCR Setup Questions, Lot Numbers, and Sample

ID’s. Open fields for “Notes”, and fields for “Email Notifications” are optional.

All user entries will be captured within the summary of the NGS Express automated reports.

ErrorChecking:RequiredEntriesQuestion: Valid Entry Invalid Entry

User ID Any selection from drop down menu

Blank (Empty)

Note: Red Error Indicator in upper right

corner of user entry field

Number of

Samples (1‐8)

Any integer value 1‐8 Blank (Empty); Any integer value 0, or

>8; Any non‐integer value



Reaction

Volume (qPCR)

Any selection from drop down menu

N/A; Forced drop down menu


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Question: Valid Entry Invalid Entry

Dilution Range

(qPCR)

Any selection from drop down menu


Notes: Any text entry

N/A (Optional Entry)

Kit Type Any selection from drop down menu


KAPA qPCR Kit

(Lot Number)

Any text or numeric value containing at

least 1 character

Blank (Empty)




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Question: Valid Entry Invalid Entry

ROX Dye (Lot

Number)

Any text or numeric value

Blank (Empty)



DNA Standards

(Lot Number)

Any text or numeric value

Blank (Empty)



Sample ID

(Unique ID’s

Required)

Any unique text or numeric value

Blank (Empty); Skipped Samples;

Duplicate Samples



Email

Message/Email

Recipients 1‐3

Any text entry

N/A (Optional Entries)


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ErrorChecking:SampleCountNGS Express Software will automatically verify DNA Library Sample counts at the start of each KAPA

qPCR Library Quantification run. Two independent user entries in the JAA “Select” tab will be compared

(Number of Samples, and the total number of Unique Sample ID’s entered). If the user entered sample

count value mismatches with the total number of Unique Sample ID’s, a User Error Message will appear.

Match (Sample Counts) Mismatch (Sample Counts)

Corrective Action: Close out of error message pop‐up window,

and return to the JAA “Select” tab. Re‐enter values for “Number

of Samples” and/or entries for “Unique Sample ID’s”.

3 ID’s 4 ID’s

Protocol

Continues

as Normal

(3 Samples Requested)


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ErrorChecking:SkippedSamplesNGS Express Software will automatically verify a continuous sequence of sample ID’s has been entered

at the start of each KAPA qPCR Library Quantification run. If blank entries are present between sample

ID’s, a User Error Message will appear.


and return to the JAA “Select” tab. Re‐enter sample unique

names for each of the “Sample ID’s” fields in continuous

sequence.

Red Tab: Entry

Error Indicator Continuous(Skipped)

Protocol

Continues

as Normal


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ErrorChecking:DuplicateSamplesNGS Express Software will automatically verify unique sample ID’s have been entered at the start of

each KAPA qPCR Library Quantification run. If duplicate entries are present, a User Error Message will

appear.


and return to the JAA “Select” tab. Re‐enter sample unique

names for each of the “Sample ID’s” fields.

Duplicate ID’s

Protocol

Continues

as Normal

Unique ID’s


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NGSExpressReportingFunctionThe Reporting function captures information about the run such as the values of runtime variables and

copies of relevant files. Upon run completion, the Reporting function will create a Zip folder located in

the C:Packard/Janus/Bin/NGSReports folder. NGS Express will create a Pipetting Summary, Run Report,

and Pipetting Log. Several properties of the run are always reported, including protocol name, test ID

(from WinPREP database), and WinPREP software version. Refer to the NGS User Installation Guide for

custom setup instructions.

NGSExpressEmailFunctionThe Email functions for KAPA qPCR Library Quantification requires user configuration for email address

and network connections. No error will message will be presented if emails are not configured or if the

network loses connectivity. Refer to the NGS User Installation Guide for custom setup instructions.