KAPA qpcr Library Quantification for NGS Express

KAPA qpcr Library Quantification for NGS Express Application Guide PerkinElmer 2200 Warrenville Road Downers Grove, IL 60515 info@perkinelmer.com 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.

Table of Contents 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 PerkinElmer 3

Required Materials List Materials Required but Not Supplied: Required Kits & Reagents Item (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 SYBR FAST ABI Prism qpcr Kit 1 x 1ml Illumina Primer Premix (10X) 6 x 80ul Illumina DNA Standards KAPA SYBR FAST Bio Rad icycler qpcr Kit 1 x 1ml Illumina Primer Premix (10X) 6 x 80ul Illumina DNA Standards KAPA SYBR FAST LightCycler qpcr Kit 1 x 1ml Illumina Primer Premix (10X) 6 x 80ul Illumina DNA Standards Water, molecular biology grade (Used for qpcr Water Controls) 10mM Tris HCL, ph 8.0 +0.05% Tween 20 (Used for Serial Dilution Buffer) MicroAmp Optical Adhesive Film (1 Film) KAPA Biosystems KK4824 (KK4601+ KK4808) KAPA Biosystems KK4835 (KK4606 + KK4808) KAPA Biosystems KK4844 (KK4607 + KK4808) KAPA Biosystems KK4854 (KK4610 + KK4808) general lab supplier general lab supplier Life Technologies 4311971 PerkinElmer 4

Labware & Consumables These materials are included in the NGS Express system package. Item (Labware & Consumables) Disposable Tip 25ul, Clear, Sterile Filter Tips, (4 Boxes) Vendor/Part Number 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 PerkinElmer 5

Custom Deck Tiles and Racks These materials are included in the NGS Express system package. Item (NGS Supports & Deck Tiles) NGS Reagent Rack (1 Rack) Vendor/Part Number 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 PerkinElmer 6

Reagent and Sample Preparation 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 preprogrammed 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. NGS Express_DNA Library Samples: 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) NGS Express_KAPA qpcr Reagents: 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) H 2 0/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 (H 2 0; 2 tubes @ 30µl) Optional Configuration: Replace one water control with an internal/custom laboratory specific control sample (H 2 0; 1 tube @ 30µl, Control; 1 tube @ 30µl) PerkinElmer 7

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 Illumina Primer Premix (10X) Volume 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 (H 2 0; 2 tubes @ 250µl) Optional Dilution Reagent: 10mM Tris HCL, ph 8.0 +0.05% Tween 20 (Tris HCL Tween; 4 tubes @ 250µl) NGS Express Well Maps The following pages show the placement of reagents and samples to start the KAPA qpcr run. PerkinElmer 8

KAPA qpcr Sample Plate Well Map 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 PerkinElmer 9

KAPA qpcr Reagent Rack Well Map 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) KAPA qpcr MMX (250µl) DNA Standard_3 (30µl) DNA Standard_4 (30µl) DNA Standard_5 (30µl) Dilution Buffer (250µl) DNA Standard_6 (30µl) H20/Control (30µl) PerkinElmer 10

KAPA qpcr Dilution Plate Well Map 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 PerkinElmer 11

KAPA qpcr Assay Plate Well Map 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. PerkinElmer 12

Advanced Protocol Logic: Validation of User Entries 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. Error Checking: Required Entries Question: 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 Note: Red Error Indicator in upper right corner of user entry field N/A; Forced drop down menu PerkinElmer 13

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 PerkinElmer 14

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 Note: Red Error Indicator in upper right corner of user entry field Blank (Empty) Sample ID (Unique ID s Required) Any unique text or numeric value Note: Red Error Indicator in upper right corner of user entry field Blank (Empty); Skipped Samples; Duplicate Samples Email Message/Email Recipients 1 3 Any text entry Note: Red Error Indicator in upper right corner of user entry field N/A (Optional Entries) PerkinElmer 15

Error Checking: Sample Count NGS 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. (3 Samples Requested) Match (Sample Counts) Mismatch (Sample Counts) 3 ID s 4 ID s Protocol Continues as Normal 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. PerkinElmer 16

Error Checking: Skipped Samples NGS 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. Continuous (Skipped) Red Tab: Entry Error Indicator Protocol Continues as Normal 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. PerkinElmer 17

Error Checking: Duplicate Samples NGS 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. Unique ID s Duplicate ID s Protocol Continues as Normal 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. PerkinElmer 18

NGS Express Reporting Function The 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. NGS Express Email Function The 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. PerkinElmer 19