KAPA qPCR Library Quantification for NGS Express · Application Guide- KAPA qPCR Library...
Transcript of KAPA qPCR Library Quantification for NGS Express · Application Guide- KAPA qPCR Library...
KAPA qPCR Library Quantification
for NGS Express
Application Guide
PerkinElmer 2200 Warrenville Road
Downers Grove, IL 60515
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.
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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
1 x 1ml Illumina Primer Premix (10X)
6 x 80ul Illumina DNA Standards
KAPA Biosystems
KK4835
(KK4606 + KK4808)
KAPA SYBR® FAST Bio‐Rad iCycler qPCR Kit
1 x 1ml Illumina Primer Premix (10X)
6 x 80ul Illumina DNA Standards
KAPA Biosystems
KK4844
(KK4607 + KK4808)
KAPA SYBR® FAST LightCycler qPCR Kit
1 x 1ml Illumina Primer Premix (10X)
6 x 80ul Illumina DNA Standards
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)
DNA Standard_2 (30µl)
DNA Standard_3 (30µl)
DNA Standard_4 (30µl)
DNA Standard_5 (30µl)
DNA Standard_6 (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
Note: Red Error Indicator in upper right
corner of user entry field
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
N/A; Forced drop down menu
Notes: Any text entry
N/A (Optional Entry)
Kit Type Any selection from drop down menu
N/A; Forced drop down menu
KAPA qPCR Kit
(Lot Number)
Any text or numeric value containing at
least 1 character
Blank (Empty)
Note: Red Error Indicator in upper right
corner of user entry field
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Question: Valid Entry Invalid Entry
ROX Dye (Lot
Number)
Any text or numeric value
Blank (Empty)
Note: Red Error Indicator in upper right
corner of user entry field
DNA Standards
(Lot Number)
Any text or numeric value
Blank (Empty)
Note: Red Error Indicator in upper right
corner of user entry field
Sample ID
(Unique ID’s
Required)
Any unique text or numeric value
Blank (Empty); Skipped Samples;
Duplicate Samples
Note: Red Error Indicator in upper right
corner of user entry field
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.
Corrective Action: Close out of error message pop‐up window,
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.
Corrective Action: Close out of error message pop‐up window,
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.