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Access Array System for the Ion Torrent PGM Sequencing System: User Guide

17

Chapter 3: Target-Specific Primer 

Validation for 4-Primer Amplicon Tagging 

on the LP 48.48 IFC

This chapter describes the validation procedure for the tagged TS primers designed in 
Chapter 2.

IMPORTANT

Access Array

™

 Barcode 1 primers for the Ion 

Torrent™ PGM™ Sequencer - 96 

must 

be ordered prior to primer validation. See Table 1 for oligo sequences and order through an 

oligonucleotide vendor.

Reference Documents

Agilent®

 DNA 1000 Kit Guide

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).

•

Store reagents according to manufacturer's storage recommendations as soon as they are 

received.

Reagents Supplied by Fluidigm

Product Name

Manufacturer

Part Number

20X Access Array Loading Reagent

Fluidigm

100-0883

Access Array Barcode 1 primers for Ion Torrent with CS1/CS2 tags

Fluidigm

—

Target-specific primer pairs tagged with universal tags (CS1 forward tag, 
CS2 reverse tag) 
50 

μ

M CS1–tagged TS forward primer 

50 

μ

M CS2–tagged TS reverse primer

Fluidigm

—

Содержание Access Array

Страница 1: ...PN 100 5024 E2 Access Array System for the Ion Torrent PGM Sequencing System USER GUIDE For Use Only with Access Array Reagents...

Страница 2: ...Fluidigm products are covered by issued and pending patents in the United States and other countries Patent and limited license information is available at fluidigm com legalnotices Limited Use Licens...

Страница 3: ...22 Required Reagents 22 Required Equipment 22 Sample Quantitation 23 Sample Normalization 23 Chapter 5 Bidirectional Amplicon Tagging on the LP 48 48 IFC 24 Reference Documents 25 Materials 25 Requir...

Страница 4: ...4 Appendix A Electropherogram Examples 41 Appendix B Access Array Barcode Library for the Ion Torrent PGM Sequencer 96 43 Appendix C Related Documents 47 Appendix D Safety 48 General Safety 48 Instrum...

Страница 5: ...uments For hazards associated with instruments this document uses indicators that include a pictogram and signal words that indicate the severity level Indicator Description Pictogram see example cons...

Страница 6: ...s To obtain SDSs for chemicals ordered from Fluidigm either alone or as part of this system go to fluidigm com sds and search for the SDS using either the product name or the part number Some chemical...

Страница 7: ...ing sequence tags and sample barcodes and 5 qualifying and quantifying harvested PCR products for sequencing The chapters in this user guide detail this workflow chronologically This Access Array targ...

Страница 8: ...mage shows a 3 x 3 grid of reaction wells containing the target specific primers and samples before reagents are loaded into the LP 48 48 IFC for library preparation prior to sequencing on the Ion Tor...

Страница 9: ...quencing System User Guide Chapter 1 Access Array System Overview 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 uniq...

Страница 10: ...are as follows Access Array System Components The Access Array system consists of the following components IFCs for Access Array LP 48 48 IFC Access Array 48 48 IFC IFC Controller AX 2 quantity for p...

Страница 11: ...ss Array 48 48 IFC enables target enrichment of 48 unique samples at the same time Specifications Primer inlets Sample inlets Control Line Fluid H1 H2 H3 H4 well well well well A1 Interface accumulato...

Страница 12: ...with Advanta NGS Library Reagent Kits see the Advanta NGS Library Preparation with Access Array Protocol PN 101 7885 Store reagents according to manufacturer s storage recommendations as soon as they...

Страница 13: ...his reagent is only required when using the Access Array 48 48 IFC and it is not for use with the LP 48 48 IFC Fluidigm 100 7966 Access Array Barcode Library for Ion Torrent PGM Sequencer 96 Fluidigm...

Страница 14: ...trifuge tubes 1 5 mL MLS 96 and 384 well PCR plates MLS Adhesive seals for PCR plates MLS P2 P1000 pipette tips Rainin Product Name Manufacturer Part Number IFC Controller AX 2 quantity pre PCR and po...

Страница 15: ...PCR activities Dedicate laboratory materials to designated areas Unless otherwise specified thaw reagents at room temperature 15 30 C and then use them at room temperature Retrieve only the reagents r...

Страница 16: ...an genomic DNA Uniplex primers are provided in nuclease free water at a final volume of 100 L per well in a single 96 well plate with mixed forward and reverse primers at a final concentration of 50 M...

Страница 17: ...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 Advan...

Страница 18: ...um of 20 seconds and centrifuge for 30 seconds NOTE The final tagged TS forward and reverse primer concentrations are 250 nM in the 5X primer solution The final TS forward and reverse primer concentra...

Страница 19: ...Scale up appropriately if a higher number of primer pairs are to be evaluated Component Volume L Final Concentration M 100 M A_BC1_CS1 forward primer 2 0 2 100 M P1_CS2 reverse primer 2 0 2 PCR Certif...

Страница 20: ...imer solution The total PCR reaction volume is 5 L 2 Vortex the 384 well PCR reaction plate for a minimum of 20 seconds and centrifuge for 30 seconds Run the PCR Reactions 1 Load the 384 well plate on...

Страница 21: ...ed above Follow the Agilent DNA 1000 Kit Guide for details On target products should account for a minimum of 50 of the total yield by mass produced for a particular primer pair If on target products...

Страница 22: ...e Quant iT PicoGreen fluorescent assay requires 1 L of sample DNA to determine sample concentration Reference Documents Quant iT PicoGreen User Guide Materials Required Reagents IMPORTANT Store reagen...

Страница 23: ...nd concentrating the sample before amplification takes place on the Access Array If the sample concentration is above 50 ng L we recommend diluting the sample to 50 ng L using DNA Suspension Buffer be...

Страница 24: ...he 3 end of the target region Set B Because both PCR products are clonally amplified onto the Ion Sphere Particle ISP at the same time one single read sequencing run will yield sequence information fo...

Страница 25: ...000 Kit Guide Materials Required Reagents Stored at 20 C FastStart High Fidelity PCR System dNTPack Sigma Aldrich PN 04738292001 20X Access Array Loading Reagent Fluidigm PN 100 0883 1X Access Array H...

Страница 26: ...ry for Ion Torrent PGM Sequencer 96 Fluidigm PN 100 4911 Target specific primer pair with universal tags CS1 forward CS2 reverse supplied in separate forward and reverse primer pools in 96 well plates...

Страница 27: ...ad of 1X Access Array Harvest Solution in the H4 well Hydration Reagent v2 ensures uniform harvest volumes when working with the Access Array 48 48 IFC but it is not needed when working with the LP 48...

Страница 28: ...onds and centrifuge for 30 seconds NOTE The final tagged TS forward and reverse primer concentrations are 4 M in the 20X primer solution The final TS forward and reverse primer concentrations in the A...

Страница 29: ...e sample pre mix for a minimum of 20 seconds and centrifuge for 30 seconds Prepare the Sample Mix Solutions 1 Combine the components listed below in a 96 well plate to prepare 48 individual sample mix...

Страница 30: ...d the LP 48 48 IFC for Access Array Select Load Mix 155x and Run Script 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 f...

Страница 31: ...n into each of the H1 H4 wells Do not use Hydration Reagent v2 here NOTE 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 H...

Страница 32: ...sferred from the IFC to the 96 well plate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 1 2 3 4 5 6 7 8 9 10 11...

Страница 33: ...100 Fold Dilution of the Harvested PCR Products 1 In a 96 well plate pipet 99 L PCR Certified Water into 48 wells 2 Add 1 L of PCR product from each sample harvested from the IFC to a separate well in...

Страница 34: ...ex the sample mix solutions for a minimum of 20 seconds and centrifuge for 30 seconds Thermal Cycle the 96 Well PCR Plate Place the PCR plates on two PCR thermal cyclers and run the following PCR prot...

Страница 35: ...ent DNA 1000 Kit Guide for details 2 Check the results of the chip to determine if the PCR product pool has the expected size Depending on the expected sizes of the PCR products a smear may be visible...

Страница 36: ...nalyzed using an Agilent 2100 Bioanalyzer to check for quality Next the PCR products are pooled together in equal volume to create one PCR product library The PCR product library is then purified usin...

Страница 37: ...f 50 130 bp is less than 25 based on the Bioanalyzer quantification shown in Appendix A Electropherogram Examples 2 Continue with the purification procedure below for all of the pooled PCR products th...

Страница 38: ...ving the tube on the bench Make sure the tube is completely dry before proceeding 16 Add 40 L of DNA suspension buffer to the microtube and vortex for 5 seconds 17 Place the microtube onto a magnetic...

Страница 39: ...ufacturer s instructions Independent IFC libraries should be quantitated separately After quantitation libraries must be normalized and volumetrically combined into a single final library for sequenci...

Страница 40: ...antify the pooled purified library by calculating the PCR Product Library Concentration outlined later in this chapter 4 Once each IFC Library has been purified and quantified normalize the concentrat...

Страница 41: ...m an Agilent 2100 Bioanalyzer electropherogram of a harvested PCR product pool The first two figures show a DNA 1000 Chip electropherogram of a pooled PCR product with 48 amplicons ranging between 180...

Страница 42: ...ncing System User Guide Appendix A Electropherogram Examples 42 The following figure shows a DNA 1000 Chip electropherogram overlay of an unpurified pooled PCR product library and the same purified po...

Страница 43: ...code Library for Ion Torrent PGM Sequencer 96 Fluidigm PN 100 4911 PN Plate Number Reagent Description Volume L 100 4912 A1 Access Array Barcode Library for Ion Torrent PGM Sequencer P1_CS2 A_BC_CS1_1...

Страница 44: ...B1 MID 26 ACATACGCGT H3 A1 B1 MID 27 ACGCGAGTAT A4 A1 B1 MID 28 ACTACTATGT B4 A1 B1 MID 29 ACTGTACAGT C4 A1 B1 MID 30 AGACTATACT D4 A1 B1 MID 31 AGCGTCGTCT E4 A1 B1 MID 32 AGTACGCTAT F4 A1 B1 MID 33...

Страница 45: ...1 MID 57 CGCGTATACA G7 A1 B1 MID 58 CGTACAGTCA H7 A1 B1 MID 59 CGTACTCAGA A8 A1 B1 MID 60 CTACGCTCTA B8 A1 B1 MID 61 CTATAGCGTA C8 A1 B1 MID 62 TACGTCATCA D8 A1 B1 MID 63 TAGTCGCATA E8 A1 B1 MID 64 TA...

Страница 46: ...D 83 AGTAGTGATC A11 A1 B1 MID 84 AGTGACACAC B11 A1 B1 MID 85 AGTGTATGTC C11 A1 B1 MID 86 ATAGATAGAC D11 A1 B1 MID 87 ATATAGTCGC E11 A1 B1 MID 88 ATCTACTGAC F11 A1 B1 MID 89 CACGTAGATC G11 A1 B1 MID 90...

Страница 47: ...95 4 Primer Amplicon Tagging with the LP 48 48 IFC 68000161 2 Primer Amplicon Tagging with the LP 48 48 IFC 68000148 Access Array Generate Tagged Primers an Excel Workbook 100 3873 Advanta NGS Library...

Страница 48: ...may create a safety hazard WARNING BIOHAZARD If you are putting biohazardous material on the instrument use appropriate personal protective equipment and adhere to Biosafety in Microbiological and Bio...

Страница 49: ...user guide for further information Hot surface hazard Do not touch potential for personal injury Biohazard Electricity hazard Indicates high electricity levels and a threat of electric shock from mac...

Страница 50: ...s protective covers No internal components are serviceable by the user WARNING ELECTRICAL HAZARD Plug the instrument into a properly grounded receptacle with adequate current capacity Chemical Safety...

Страница 51: ...For technical support visit fluidigm com support...

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