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Summary of Contents for Fluidigm Access Array
- Page 1 PN 100-5024 E2 Access Array System for the Ion Torrent PGM Sequencing System USER GUIDE For Use Only with Access Array Reagents...
- Page 2 © 2018 Fluidigm Corporation. All rights reserved. 06/2018 For technical support visit fluidigm.com/support. North America +1 650 266 6100 | Toll-free (US/CAN): 866 358 4354 | techsupport@fluidigm.com Latin America +1 650 266 6100 | techsupportlatam@fluidigm.com Europe/Middle East/Africa/Russia +44 1223 598100 | techsupporteurope@fluidigm.com Japan +81 3 3662 2150 | techsupportjapan@fluidigm.com...
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Page 3: Table Of Contents
Normalization PCR Product Library Quantitation Procedure Reference Documents Agilent 2100 Bioanalyzer Quantification PicoGreen Fluorimetry Quantification Materials Pool the Products from Multiple Access Array Required Reagents IFCs Required Equipment Access Array System for the Ion Torrent PGM Sequencing System: User Guide... - Page 4 Library for the Ion Torrent PGM Sequencer - 96 Appendix C: Related Documents Appendix D: Safety General Safety Instrument Safety Symbols on the Instrument Electrical Safety Chemical Safety Access Array System for the Ion Torrent PGM Sequencing System: User Guide...
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Page 5: About This Guide
Failure to follow these guidelines may result in undesirable effects, injury to personnel, and/or damage to the instrument or to property. • This guide is for preparing sequencing libraries with Access Array™ reagents. To prepare targeted DNA sequencing libraries with Advanta™ NGS Library Reagent Kits, see the Advanta NGS Library Preparation with Access Array Protocol (PN 101-7885). -
Page 6: Safety Data Sheets
Safety Data Sheets Read and understand the SDSs before handling chemicals. To obtain SDSs for chemicals ordered from Fluidigm, either alone or as part of this system, go to fluidigm.com/sds search for the SDS using either the product name or the part number. - Page 7 2) quantifying samples, 3) running an integrated fluidic circuit (IFC, either the LP 48.48 IFC or the Access Array 48.48 IFC), 4) attaching sequence tags and sample barcodes, and 5) qualifying and quantifying harvested PCR products for sequencing.
- Page 8 Ion Torrent™ PGM™ Sequencer - 96 First, TS forward and reverse primers are loaded into each column of the reaction wells on the LP 48.48 IFC. Access Array System for the Ion Torrent PGM Sequencing System: User Guide...
- Page 9 Samples are then loaded into each row of reaction wells on the LP 48.48 IFC: After loading the IFC, each reaction well contains a unique combination of TS primer pairs and samples: Access Array System for the Ion Torrent PGM Sequencing System: User Guide...
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Page 10: Chapter 1: Access Array System Overview Access Array System Components
FC1™ cycler IFC Controller AX FC1 cycler IFC Controller AX Pre-PCR Post-PCR NOTE The stand-alone thermal cycler has been discontinued. It has been replaced by the FC1 cycler. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 11: Access Array Ifcs
Sample inlets well well Containment accumulator Control Line Fluid The high-throughput LP 48.48 IFC (or Access Array 48.48 IFC) enables target enrichment of 48 unique samples at the same time. Specifications Description Parameter Footprint dimensions 128 x 85 x 14 mm Inlet spacing on input frame 4.5 mm pitch... -
Page 12: Access Array System Workflow
The Access Array system workflow consists of five major steps: Workflow Step Dispense Transfer samples and primers to the inlets on the LP 48.48 IFC (or Access Array 48.48 IFC) from a standard 96-well plate, using an 8-channel pipette. Load... -
Page 13: Required Consumables
It has been replaced by Room temperature the LP 48.48 IFC. LP Control Line Fluid 48.48 or Fluidigm 101-2345 Control Line Fluid for 48.48 IFCs 89000020 Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 14: Required Equipment
Single-channel P2-P1000 pipettes Rainin — 8-channel P20 pipette Rainin — Example Sample and Primer Layout in 96-Well Plates For ease of pipetting, fill the 96-well plates by columns, not rows. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 15: Before You Begin
This protocols in this document require the use of updated scripts for FC1 (v1.6 or later) and IFC Controller AX (v2.8 or later) that can be downloaded from fluidigm.com/software. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 16: Chapter 2: Designing Targeted Sequencing Primers
Chapter 2: Designing Targeted Sequencing Primers You can design your own Access Array target-specific primers using available online tools (for example, UCSC website at genome.ucsc.edu/cgi-bin/hgGateway, Primer3 website at bioinfo.ut.ee/primer3/, and in silico PCR at genome.ucsc.edu/cgi-bin/ hgPcr?hgsid=136961442) or by using the D3™... -
Page 17: Chapter 3: Target-Specific Primer Validation For 4-Primer Amplicon Tagging On The Lp 48.48
Materials Required Reagents IMPORTANT • This guide is for preparing sequencing libraries with Access Array reagents. To prepare targeted DNA sequencing libraries with Advanta NGS Library Reagent Kits, see the Advanta NGS Library Preparation with Access Array Protocol (PN 101-7885). -
Page 18: Prepare Primer Validation Reactions
The primer validation protocol prepares enough reagents to perform 48 primer validation reactions. IMPORTANT It is essential to have Access Array Barcode 1 primers before proceeding with the primer validation. See Table 1 below for the oligo sequences. Table 1. Access Array Barcode 1 primers for Ion Torrent Name Sequence (5′-3′) - Page 19 Table 4 displays the volumes required to prepare 48 reactions with a 25% overage to evaluate 48 target-specific primer pairs. Scale up appropriately if a higher number of primer pairs are to be evaluated. Access Array System for the Ion Torrent PGM Sequencing System: User Guide...
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Page 20: Set Up The Pcr Reactions In A 384-Well Pcr Plate
95 ºC 15 sec 60 ºC 30 sec 72 ºC 1 min 95 ºC 15 sec 80 ºC 30 sec 60 ºC 30 sec 72 ºC 1 min Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 21: Check Pcr Products On The Agilent 2100 Bioanalyzer
Figure 1. Example of 48 primer pairs checked on an Agilent chip. The red-labeled PCR reactions generated multiple or incorrectly sized primers, which need to be redesigned and validated before continuing. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 22: Chapter 4: Sample Quantitation And Normalization
Part Number Quant-iT PicoGreen dsDNA Assay Kit Thermo Fisher Scientific P11496 Required Equipment Product Name Manufacturer Part Number Fluorimeter-compatible 96- or 384-well microtiter plates Major laboratory supplier — (MLS) Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 23: Sample Quantitation
Therefore: Dilute 10 μL of the 200 ng/μL sample in 30 μL of DNA Suspension Buffer to obtain a 50 ng/μL sample concentration. • Normalize all sample concentrations to 50 ng/μL. Samples are now ready for amplification using the LP 48.48 IFC. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 24: Chapter 5: Bidirectional Amplicon Tagging On The Lp 48.48
This protocol outlines the bidirectional amplicon tagging strategy for the Ion Torrent™ PGM™ for amplicon libraries that have been generated on the Access Array™ system. Sequencer - 96 The goal of this protocol is to sequence both ends of an amplicon with a single-read sequencing run. -
Page 25: Reference Documents
1X Access Array Harvest Solution (Fluidigm, PN 100-1031) is not packaged for individual sale. It can be purchased in units of 10, under the name Access Array Harvest Pack, PN 100-3155, or as a component in the 48.48 Access Array Loading Reagent Kit, PN 100-1032. -
Page 26: Prime The Ifc
1X Access Array Hydration Reagent v2 (Fluidigm, PN 100-7966) IMPORTANT This reagent is not required, but it ensures uniform harvest volumes if used priming step when using the Access Array 48.48 IFC. (It is not for use with during the the LP 48.48 IFC.) •... - Page 27 Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC Prime the IFC IMPORTANT • Use the LP 48.48 IFC (or the Access Array 48.48 IFC) within 24 hours of opening the package. • Due to different accumulator volumes, use only 48.48 syringes with 300 μL of control line fluid (PN 89000020).
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Page 28: Prepare The 20X Primer Solutions
Prepare the 20X Primer Solutions NOTE If you are using primers provided by the Fluidigm Assay Design Group, refer to the order informatics packet for correct primer concentrations. Prepare the 20X primer solutions for 48 individual primer pairs as shown in the table below. -
Page 29: Prepare Sample Pre-Mix And Samples
Combine the components listed below in a 96-well plate to prepare 48 individual sample mix solutions. Table 9. Sample mix solution Component Volume (μL) Final Concentration Sample pre-mix genomic DNA (50 ng/μL) 10 ng/μL Total Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 30: Load The Ifc
NOTE For Access Array 48.48 IFCs, select Load Mix v7 (151x) and Run Script. Load Mix v7 (151x) is a script update from Load Mix (151x) for Access Array 48.48 IFCs and can be downloaded from fluidigm.com/software. Contact Technical Support for assistance. -
Page 31: Thermal-Cycle The Ifc
If you are working with the LP 48.48 IFC or the Access Array 48.48 IFC, use 1X Access Array Harvest Solution in all four wells (H1-H4). Pipet 2 μL of 1X Access Array Harvest Solution into each of the sample inlets on the IFC. Load the IFC into the Post-PCR IFC Controller AX located in the Post-PCR lab. - Page 32 Harvest the IFC Sample inlets with DO NOT USE harvested samples The following image displays a map of the PCR products when transferred from the IFC to the 96-well plate: Access Array System for the Ion Torrent PGM Sequencing System: User Guide...
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Page 33: Attach Sequence Tags And Sample Barcodes
Vortex the PCR product dilutions for a minimum of 20 seconds and centrifuge for 30 seconds. Prepare the Sample Mix Solutions Combine the components listed below in two 96-well plates to prepare two sets of 48 individual sample mix solutions. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 34: Thermal-Cycle The 96-Well Pcr Plate
Table 13. PCR protocol for attaching sequence tags and sample barcodes PCR Stages Cycle 95°C 10 min 95°C 15 sec 60°C 30 sec 72°C 1 min 72°C 3 min Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 35: Check Pcr Products On The Agilent 2100 Bioanalyzer
+63 bp when compared to Harvest Pool Products. Store the PCR products at –20 ºC. Next Step Continue with your library preparation as described in Post-PCR Amplicon Purification and Quantitation on page Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 36: Chapter 6: Post-Pcr Amplicon Purification And Quantitation
This chapter describes a standard Access Array™ procedure for the analysis of PCR products harvested from an LP 48.48 IFC. The quality of the PCR products prepared on an LP 48.48 IFC (or a Access Array 48.48 IFC) is critical for successful amplicon sequencing. Any contamination of primers or primer dimers in the PCR products will be directly reflected in the quality of sequencing reads. -
Page 37: Quantitate Pcr Products
Pipet the barcoded sample pool, DNA suspension buffer, and AMPure XP beads into a 1.5 mL microtube according to the table below: Component Volume (μL) 12.0 Barcoded sample pool DNA suspension buffer 24.0 36.0 AMPure XP Beads 72.0 Total Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 38: Pcr Product Library Quantitation Procedure
Select the Region Table subtab on the bottom panel of the Electropherogram tab. Right-click the electropherogram and select Add region. Define the region to cover all of the PCR product library peaks. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 39: Picogreen Fluorimetry Quantification
Quant-iT is then used for the downstream sequencing preparation. Pool the Products from Multiple Access Array IFCs To pool amplicons from more than one Access Array IFC, pool and purify each Access Array IFC separately. Follow the steps outlined in this chapter under... - Page 40 IFC Library and pool libraries volumetrically by adding an equal volume of each library to a new microcentrifuge tube. The pooled Access Array IFC library is now ready for sequencing. Access Array System for the Ion Torrent PGM Sequencing System: User Guide...
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Page 41: Appendix A: Electropherogram Examples
Chip electropherogram of a pooled PCR product with 48 amplicons ranging between 180– 350 bp. Figure A shows the harvested PCR product pool from a Access Array 48.48 IFC, and Figure B shows the same harvested PCR product pool after SPRI cleanup:... - Page 42 The following figure shows a DNA 1000 Chip electropherogram overlay of an unpurified pooled PCR product library and the same purified pooled PCR product library after AMPure XP cleanup: Access Array System for the Ion Torrent PGM Sequencing System: User Guide...
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Page 43: Appendix B: Access Array Barcode Library For The Ion Torrent Pgm Sequencer
100-4913 Access Array Barcode Library for Ion Torrent PGM Sequencer P1_CS1 / A_BC_CS2_1-96 at 2 μM per oligo Table 15. Barcode Sequences for Access Array Barcode Library for Ion Torrent PGM Sequencer - 96 (Fluidigm, PN 100-4911) Barcode Name Barcode Sequence... - Page 44 Appendix B: Access Array Barcode Library for the Ion Torrent PGM Sequencer - 96 Barcode Name Barcode Sequence Barcode Well Plate MID-16 TCACGTACTA A1 & B1 MID-17 CGTCTAGTAC A1 & B 1 MID-18 TCTACGTAGC A1 & B1 MID-19 TGTACTACTC A1 & B1...
- Page 45 Appendix B: Access Array Barcode Library for the Ion Torrent PGM Sequencer - 96 Barcode Name Barcode Sequence Barcode Well Plate MID-47 TGTGAGTAGT A1 & B1 MID-48 ACAGTATATA A1 & B1 MID-49 ACGCGATCGA A1 & B1 MID-50 ACTAGCAGTA A1 & B1...
- Page 46 Appendix B: Access Array Barcode Library for the Ion Torrent PGM Sequencer - 96 Barcode Name Barcode Sequence Barcode Well Plate MID-78 ACGTCTCATC A1 & B1 MID-79 ACTCATCTAC A1 & B1 MID-80 ACTCGCGCAC A1 & B1 MID-81 AGAGCGTCAC A1 & B1...
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Page 47: Appendix C: Related Documents
– Generate Tagged Primers (an Excel Workbook) 100-3873 Advanta NGS Library Preparation with Access Array Protocol 101-7885 Advanta NGS Library Preparation on the LP 48.48 IFC with Access Array Quick 101-7886 Reference IFC Controller AX User Guide 68000157 FC1™ Cycler User Guide... -
Page 48: Appendix D: Safety
CAUTION PINCH HAZARD. The instrument door and tray can pinch your hand. Make sure your fingers, hands, and shirtsleeves are clear of the door and tray when loading or ejecting an integrated fluidic circuit (IFC). Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 49: Symbols On The Instrument
To minimize negative environmental impact from disposal of electronic waste, do not dispose of electronic waste in unsorted municipal waste. Follow local municipal waste ordinances for proper disposal provision. Contact customer service for information about responsible disposal options. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... -
Page 50: Electrical Safety
When working with any chemicals, refer to the applicable safety data sheets (SDSs) provided by the manufacturer or supplier. Access Array System for the Ion Torrent PGM Sequencing System: User Guide... - Page 51 For technical support visit fluidigm.com/support.