Ion AmpliSeq Exome RDY Library Preparation

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1 USER GUIDE Ion AmpliSeq Exome RDY Library Preparation for use with: Ion AmpliSeq Exome RDY Kit Ion AmpliSeq Exome S5 RDY Kit Catalog Numbers A27192, A27193, A29854, and A29855 Publication number MAN Revision C.0 For Research Use Only. Not for use in diagnostic procedures.

2 The information in this document is subject to change without notice. DISCLAIMER TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Trademarks All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc. Agencourt and AMPure are trademarks of Beckman Coulter, Inc. NanoDrop is a trademark of NanoDrop Technologies, LLC Thermo Fisher Scientific Inc. All rights reserved. 2 Ion AmpliSeq Exome RDY Library Preparation User Guide

3 Contents About This Guide Changes from previous version Purpose of this guide CHAPTER 1 Product information Product description Ion AmpliSeq Exome and Exome S5 RDY Kits Shared Kit Components Kit-specific components (Optional) Ion Library Equalizer Kit (Required) Ion Xpress Barcode Adapters Required materials and equipment Procedure overview Procedure guidelines CHAPTER 2 Methods Workflow Input DNA requirements Quantify DNA Amount and volume of DNA needed per target amplification reaction Amplify targets Combine PCR products Partially digest primer sequences Ligate barcode adapters to the amplicons and purify Barcoded libraries only: Combine and dilute adapters Set up and run the ligation reaction Purify the unamplified library Option 1: Equalize the library Before starting Amplify the library Wash the Equalizer Beads Add Equalizer Capture to the amplified library Add the Equalizer Beads and wash Elute the equalized library (Optional) Combine equalized libraries Ion AmpliSeq Exome RDY Library Preparation User Guide 3 CONFIDENTIAL For Developer Access Use Only Do Not Distribute

4 Contents Store equalized libraries Option 2: Quantify the unamplified library by qpcr Elute the unamplified library Quantify the library by qpcr and calculate the dilution factor (Optional) Combine amplicon libraries Store libraries Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument Amplify the library Purify the amplified library Qubit 2.0 Fluorometer: Quantify the library and calculate the dilution factor Agilent Bioanalyzer : Quantify the library and calculate the dilution factor (Optional) Combine amplicon libraries Store libraries CHAPTER 3 Combining Ion AmpliSeq libraries Ion Chip capacities for Ion AmpliSeq libraries APPENDIX A Tips and Troubleshooting Tips and modifications to the standard workflow Tips Modifications to the standard workflow Troubleshooting APPENDIX B Supplemental information Data analysis Install the hg19 reference on your Torrent Server Import the BED files into your Torrent Server Coverage Analysis Plugin Torrent Variant Caller Plugin Copy number variation analysis using Ion Reporter Software APPENDIX C Safety Chemical safety Biological hazard safety APPENDIX D Documentation and Support Customer and technical support Limited Product Warranty Ion AmpliSeq Exome RDY Library Preparation User Guide

5 About This Guide CAUTION! ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard types specified in procedures may be abbreviated in this document. For the complete safety information, see the Safety appendix in this document. IMPORTANT! Before using this product, read and understand the information the Safety appendix in this document. Changes from previous version Revision Date Description C.0 07 September 2015 Added new Ion AmpliSeq Exome S5 RDY Kit information B.0 24 Februrary 2015 Updated product catalog numbers A.0 27 March 2014 New user guide Corrected typo in Input DNA requirements Purpose of this guide The Ion AmpliSeq Exome RDY Library Preparation Guide provides reference information for the products listed on the following page. Ion AmpliSeq Exome RDY Library Preparation User Guide 5

6 1 Product information Product description This guide covers the following products: Ion AmpliSeq Exome RDY Kit, 4x2 (Cat. no. A27193) and Ion AmpliSeq Exome RDY Kit, 1x8 (Cat. no. A27192) Ion AmpliSeq Exome S5 RDY Kit, 4x2 (Cat. no. A29855) and Ion AmpliSeq Exome RDY Kit, 1x8 (Cat. no. A29854) Ion Library Equalizer Kit (Cat. no ) Ion Xpress Barcode Adapters 1 96 Kits (various cat. nos.) Ion AmpliSeq Exome RDY Kit contains reagents for the rapid preparation of 8 exome libraries for sequencing using the Ion Proton System. This kit includes Exome RDY plates that have 12 primer pools dried into 2 rows of 4 plates (Cat. no. A27193) or 8 rows of one plate (Cat. no. A27192) to minimize pipetting steps and facilitate automation. The plates are bundled with the Ion AmpliSeq Library Kit Plus to increase uniformity, robustness, and performance with the Ion Equalizer Kit. This kit also includes the Ion PI Chip Kit v3. Ion AmpliSeq Exome S5 RDY Kit contains all of the same reagents as the Ion AmpliSeq Exome RDY Kits, but instead includes the Ion 540 Chip for use with the Ion S5 or S5 XL Systems. Ion Xpress Barcode Adapters 1 96 Kits enable the preparation of barcoded libraries using the above-mentioned library kit. Multiple barcoded libraries can be combined and loaded onto a single Ion Chip to minimize the sequencing run time and cost. Ion Library Equalizer Kit provides an optional, streamlined method for normalizing library concentration without the need for quantitation. 6 Ion AmpliSeq Exome RDY Library Preparation User Guide

7 Chapter 1 Product information Ion AmpliSeq Exome and Exome S5 RDY Kits 1 Ion AmpliSeq Exome and Exome S5 RDY Kits The Ion AmpliSeq Exome (Cat. nos. A27192 and A27193) and Exome S5 (Cat. nos. A29854 and A29855) RDY Kits provide primers, library reagents, and chips for preparing and running 8 exome libraries. The Ion AmpliSeq Exome RDY Kits are for use with the Ion Proton System, and include the Ion PI Chip Kit v3 (Cat. no. A26771). The Ion Exome S5 RDY Kits are for use with the Ion S5 or S5 XL Systems, and include the Ion 540 Chip Kit (Cat. no. A27766). Shared kit components Ion AmpliSeq Library Kit Plus Component Cap color Number of tubes Volume per tube Storage 5X Ion AmpliSeq HiFi Mix Red 1 tube 130 µl 30 C to 10 C FuPa Reagent Brown 1 tube 60 µl Switch Solution Yellow 1 tube 130 µl DNA Ligase Blue 1 tube 60 µl 25X Library Amp Primers White 1 tube 60 µl 1X Library Amp Mix Black 1 tube 1.5 ml Low TE Clear 1 tube 1.7 ml Room temp (15 C to 30 C) Ion AmpliSeq Exome RDY Panels Component Quantity Storage Ion AmpliSeq Exome RDY Panel, 1x2 4 plates (8 libraries) Room temp Ion AmpliSeq Exome RDY Panel, 1x8 1 plate (8 libraries) (15 C to 30 C) Ion AmpliSeq Exome RDY Library Preparation User Guide 7

8 1 Chapter 1 Product information (Optional) Ion Library Equalizer Kit Kit-specific components Ion PI Chip Kit v3 (included in Cat. nos. A27192 and A27193) Component Quantity Storage Ion PI Chip 8 chips Room temp (15 C to 30 C) Ion 540 Chip Kit (included in Cat. nos. A29854 and A29855) Component Quantity Storage Ion 540 Chip 8 chips Room temp (15 C to 30 C) (Optional) Ion Library Equalizer Kit The Ion Library Equalizer Kit contains reagents for 96 reactions: Ion Library Equalizer Kit (Cat. no ) Component Cap color Quantity Volume Storage Equalizer Primers Pink 1 tube 200 µl Equalizer Capture Purple 1 tube 1 ml Equalizer Elution Buffer Clear 1 bottle 10 ml Equalizer Beads Orange 1 tube 300 µl 2 C to 8 C Equalizer Wash Buffer Clear 1 bottle 35 ml Room temp (15 C to 30 C) (Required) Ion Xpress Barcode Adapters Ion Xpress Barcode Adapters Kits are required for preparing barcoded libraries. Each kit includes reagents sufficient for preparing up to 40 Ion AmpliSeq libraries per barcode (40 16 libraries). Ion Xpress Barcode Adapters 1 16 Kit (Cat. no ) Ion Xpress Barcode Adapters Kit (Cat. no ) Ion Xpress Barcode Adapters Kit (Cat. no ) Ion Xpress Barcode Adapters Kit (Cat. no ) Ion Xpress Barcode Adapters Kit (Cat. no ) Ion Xpress Barcode Adapters Kit (Cat. no ) Ion Xpress Barcode Adapters 1-96 (Cat. no ) 8 Ion AmpliSeq Exome RDY Library Preparation User Guide

9 Chapter 1 Product information Required materials and equipment 1 Required materials and equipment Unless otherwise indicated, all materials are available through thermofisher.com. MLS: Fisher Scientific ( or other major laboratory supplier. One of the following: Description GeneAmp PCR System 9700 Single or Dual 96-well Thermal Cycler AB 2720 Thermal Cycler Veriti 96-well Thermal Cycler Proflex 96-well PCR System MicroAmp Optical 96-well Reaction Plates (Optionaldepending on library preparation method) Source See web product pages N (with barcode) MicroAmp Clear Adhesive Film MicroAmp Optical Film Compression Pad Agencourt AMPure XP Kit DynaMag -96 Side, or other plate magnet Nuclease-Free Water Absolute ethanol Pipettors, μl, and low-retention filtered pipette tips Choose one option for library quantitation and normalization: Beckman Coulter, A63880 or A D AM9932 MLS MLS Ion Library Quantitation Kit Qubit 2.0 Fluorometer and Qubit dsdna HS Assay Kit Agilent 2100 Bioanalyzer instrument and Agilent High Sensitivity DNA Kit Recommended for DNA quantitation: TaqMan RNase P Detection Reagents Kit Optional for DNA quantitation: Qubit 2.0 Fluorometer and Qubit dsdna HS Assay Kit Ion Xpress Barcode Adapters (Optional): Real-time PCR instrument (e.g., Applied Biosystems 7900HT, 7500, StepOne, StepOnePlus, ViiA 7 Systems, or QuantStudio 12K Flex Real-Time PCR System) Q32866, Q32851 Agilent, G2939AA, Q32866, Q32851 See web product pages See web product pages (Optional): MagMax DNA Multi-Sample Kit (Optional): PureLink Genomic DNA Mini Kit K Ion AmpliSeq Exome RDY Library Preparation User Guide 9

10 1 Chapter 1 Product information Procedure overview Procedure overview Amplify target regions from ng of genomic DNA (gdna) in the Ion AmpliSeq Exome RDY plates and the Ion AmpliSeq HiFi Mix. Treat amplicons with FuPa Reagent to partially digest the primers and phosphorylate the amplicons. The amplicons are then ligated to Ion Xpress Barcode Adapters and purified. Libraries can be normalized without the need for quantitation or dilution using the Ion Library Equalizer Kit. Alternatively, libraries may be quantified by qpcr without amplification. If you intend to quantify libraries with a method that does not specifically detect amplifiable molecules, such as the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument, library amplification and purification are required before quantitation. Up to 3 barcoded exome libraries may be combined prior to template preparation and sequencing. 10 Ion AmpliSeq Exome RDY Library Preparation User Guide

11 Chapter 1 Product information Procedure guidelines 1 Procedure guidelines Thaw components that contain enzymes such as 5X AmpliSeq HiFi Mix, FuPa Reagent, DNA Ligase and 1X Library Amp Mix on ice, and keep on ice during the procedure. All other components may be thawed at room temperature. Gently vortex and spin down all reagents before use. If there is visible precipitate in the Switch Solution or the tube cap after thawing, vortex or pipet up and down at room temperature to resuspend. Use good laboratory practices to minimize cross-contamination of products. If possible, perform PCR setup in an area or room that is separate from template preparation. Always change pipette tips between samples. Use a calibrated thermal cycler specified in Required materials and equipment on page 9. Pipet viscous solutions slowly and ensure complete mixing by vigorous vortexing or pipetting up and down several times. Ion AmpliSeq Exome RDY Library Preparation User Guide 11

12 2 Methods Workflow Amplify targets (page 13) Genomic DNA Combine PCR products Amplify targets using Ion AmpliSeq Primer Pool Partially digest primer sequences (page 14) Partially digest primer sequences Ligate barcode adapters to the amplicons and purify (page 15) Option 1: Equalize the library (page 17) Option 2: Quantify the unamplified library by qpcr (page 20) Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument (page 22) Adapters OR Barcode Adapters A X A P1 X P1 OR Ligate adapters P1 Nonbarcoded library P1 Barcoded library Combine libraries (optional) and proceed to template preparation. 12 Ion AmpliSeq Exome RDY Library Preparation User Guide

13 Chapter 2 Methods Input DNA requirements 2 Input DNA requirements Quantify DNA Amount and volume of DNA needed per target amplification reaction We recommend the TaqMan RNase P Detection Reagents Kit (Cat. no ) for quantifying amplifiable DNA (see DOC-7431). The Qubit dsdna HS Assay Kit (Cat. no. Q32851 or Q32854) may also be used. Methods such as densitometry (e.g., NanoDrop Spectrophotometers) are not recommended, because they do not discriminate between DNA and RNA and thus are extremely sensitive to small fragments of hydrolyzed RNA. This can lead to gross overestimation of the concentration of sample DNA, underseeding of the target amplification reaction, low-quality libraries, and low library yields. For each exome library, use ng of genomic DNA (gdna). The maximum volume of DNA is 56 µl. Amplify targets 1. For each sample, prepare a master mix: Component Volume 2. Mix thoroughly. 5X Ion AmpliSeq HiFi Mix (red cap) 14 µl ng gdna (non-ffpe) * Y µl Nuclease-free Water (56 Y) µl Total 70 µl * Where DNA is not limiting, we recommend 100 ng. For library amplification followed by Qubit quantitation, we recommend 50 ng. 3. Remove the seal from the Ion AmpliSeq Exome RDY plate. 4. For each sample, use a low volume pipettor to carefully dispense 5-µL aliquots of master mix into a single row (12 wells) of the plate without changing the tip. Note: A blue dye has been added to the primers to help identify wells that contain dry primers. All rows in the 1x8 format (Part. no ) contain primers; only rows C and F in the 1x2 format (Part. no ) contain primers. 5. Apply a MicroAmp clear adhesive film (Cat. no ), ensure a tight seal, and briefly centrifuge the plate to collect droplets. 6. Place a MicroAmp Compression Pad on the plate, load the plate in the thermal cycler, and run the following program for 5 µl volume. IMPORTANT! Use of the recommended plates, seals, compression pads, and a Thermo Fisher Scientific thermal cycler are critical for best performance. Ion AmpliSeq Exome RDY Library Preparation User Guide 13

14 2 Chapter 2 Methods Combine PCR products Stage Step Temp Time Hold Activate 99 C 2 min enzyme Cycle Denature 99 C 15 sec (10 cycles) Anneal/extend 60 C 16 min Hold 10 C Hold STOPPING POINT PCR products may be stored at 10 C overnight. For longer periods, store at 20 C. Combine PCR products 1. Briefly centrifuge the plate to collect droplets. Carefully remove the plate seal and combine the 12 PCRs for each sample (row) by transferring the material from wells 1 5 and 7 12 into the column 6 well, without changing the tip. Combine PCRs Amplify Partially digest primer sequences 1. Add 6 µl of FuPa Reagent (brown cap) to each combined PCR product. The total volume is approximately 60 µl. 2. Seal the plate with MicroAmp adhesive film, vortex thoroughly, and spin down to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times prior to sealing the plate. 3. Place a MicroAmp Compression Pad on the plate, load the plate in the thermal cycler, and run the following program: Temperature Time 50 C 10 min 55 C 10 min 60 C 20 min 10 C Hold (for up to 1 hour) 14 Ion AmpliSeq Exome RDY Library Preparation User Guide

15 Chapter 2 Methods Ligate barcode adapters to the amplicons and purify 2 Ligate barcode adapters to the amplicons and purify Barcoded libraries only: Combine and dilute adapters For each barcode X chosen, prepare a mix of Ion P1 Adapter and Ion Xpress Barcode X at a final dilution of 1:4. Use 6 µl of this barcode adapter mix for the Ion AmpliSeq Adapters in step 2 below. Diluted adapters should be stored at 20 C. For example, combine the volumes indicated in the following table. Scale volumes as necessary. IMPORTANT! When handling barcoded adapters, be careful to avoid crosscontamination by changing gloves frequently and opening one tube at a time. Set up and run the ligation reaction 1. Carefully remove the plate seal and add 6 µl of diluted barcode adapters to each well of digested amplicons: Example barcode adapter mix for up to 4 reactions Component Volume Ion P1 Adapter 1.5 µl Ion Xpress Barcode X * 1.5 µl Nuclease-free Water 3 µl Total 6 µl * X = barcode chosen 2. Add 12 µl of Switch Solution to each well. If there is visible precipitate in the Switch Solution, vortex or pipet up and down at room temperature to resuspend. 3. Add 6 µl of DNA Ligase to each well to bring the total reaction volume to approximately 80 µl. Note: Do not combine DNA Ligase and barcode adapters prior to adding to reaction mixture. 4. Seal the plate with MicroAmp adhesive film, vortex thoroughly and briefly centrifuge to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times prior to sealing the plate. 5. Place a MicroAmp Compression Pad on the plate, load the plate in the thermal cycler, and run the following program: Temperature Time 22 C 30 min 72 C 10 min 10 C Hold (for up to 1 hour) STOPPING POINT Samples may be stored at 20 C. Ion AmpliSeq Exome RDY Library Preparation User Guide 15

16 2 Chapter 2 Methods Ligate barcode adapters to the amplicons and purify Purify the unamplified library IMPORTANT! Bring AMPure XP reagent to room temperature and vortex thoroughly to disperse the beads before use. Pipet solution slowly. Use freshly prepared 70% ethanol for the next steps. Combine 230 µl of ethanol with 100 µl of nuclease-free water per sample. Do NOT substitute a Dynabeads -based purification reagent for the Agencourt AMPure XP reagent. 1. Carefully remove the plate seal and add 80 µl (~1X sample volume) of Agencourt AMPure XP Reagent to each library and pipet up and down 5 times to thoroughly mix the bead suspension with the DNA. 2. Incubate the mixture for 5 minutes at room temperature. 3. Place the plate in a magnetic rack such as the DynaMag -96 Side Magnet (Cat. no D) and incubate for 2 minutes or until solution clears. Carefully remove and discard the supernatant without disturbing the pellet. 4. Add 150 µl of freshly prepared 70% ethanol and move the plate side to side in the magnet to wash the beads, then remove and discard the supernatant without disturbing the pellet. Note: If your magnet does not have two positions for shifting the beads, remove the plate from the magnet and gently pipet up and down five times (with the pipettor set at 100 µl), then return the plate to the magnet and incubate for 2 minutes or until the solution clears. 5. Repeat step 4 for a second wash. 6. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in the magnet, air-dry the beads at room temperature for 5 minutes. Do not overdry. Note: Residual ethanol drops will inhibit library amplification. If necessary, spin down the plate and remove residual ethanol, prior to air-drying the beads. 7. Proceed immediately to one of the following: Option 1: Equalize the library below or Option 2: Quantify the unamplified library by qpcr on page 20 or Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument on page 22. Note: Since qpcr typically provides the best sensitivity and barcode balance, it is the recommended quantitation method. The Ion Library Equalizer Kit offers the greatest convenience, but can result in sample loss if library yield is too low. Qubit 2.0 fluorometry is the most economical, but lacks specificity. Agilent 2100 Bioanalyzer quantitation generates the most information for troubleshooting, but lacks accuracy due to the 1 µl sample volume. 16 Ion AmpliSeq Exome RDY Library Preparation User Guide

17 Chapter 2 Methods Option 1: Equalize the library 2 Option 1: Equalize the library The Ion Library Equalizer Kit (Cat. no ) provides a method for normalizing library concentration at ~100 pm without the need for quantitation. First amplify the Ion AmpliSeq library, then capture the library on Equalizer Beads and dilute. Before starting Warm all the reagents in the Ion Library Equalizer Kit to room temperature. Vortex and briefly spin down all reagents before use. Amplify the library 1. Remove the plate from the magnet and add 50 µl of 1X Library Amp Mix and 2 µl of 25X Library Amp Primers to each bead pellet. The Library Amp Mix and Library Amp Primers may be combined before addition. Note: 25X Library Amp Primers are compatible with the Ion Library Equalizer Kit. You may use either the 25X Library AMP Primers or the Equalizer Primers to amplify the library. Note: Library amplification takes place in the presence of the AMPure XP beads. 2. Place a MicroAmp Compression Pad on the plate, load the plate in the thermal cycler, and run the following program. During cycling, wash the Equalizer Beads, if necessary. Stage Temperature Time Hold 98 C 2 min 7 cycles 98 C 15 sec 64 C 1 min Hold 10 C Hold (for up to 1 hour) Wash the Equalizer Beads Note: Beads for multiple reactions may be prepared in bulk, and can be stored in Equalizer Wash Buffer at 4 C for up to 6 months until use. After 6 months, beads should be rewashed with an equal volume of Equalizer wash buffer. 1. Bring the Equalizer Beads to room temperature and mix thoroughly. 2. For each reaction, pipet 3 µl of beads into a clean strip tube or plate well and add 6 µl/reaction of Equalizer Wash Buffer. 3. Place the strip tube/plate in a magnetic rack for 3 minutes or until the solution is completely clear. 4. Carefully remove and discard the supernatant without disturbing the pellet. 5. Remove the tube/plate from the magnet, add 6 µl/reaction of Equalizer Wash Buffer, and pipet up and down to resuspend. If not previously performed. Ion AmpliSeq Exome RDY Library Preparation User Guide 17

18 2 Chapter 2 Methods Option 1: Equalize the library Add Equalizer Capture to the amplified library 1. After thermal cycling, remove the seal from the plate and add 10 µl of Equalizer Capture to each library amplification reaction. 2. Seal the plate with MicroAmp adhesive film, vortex thoroughly, and spin down to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times prior to sealing the plate. 3. Incubate at room temperature for 5 minutes. Add the Equalizer Beads and wash 1. Mix the washed Equalizer Beads by gentle vortexing or pipetting up and down. 2. Add 6 µl of washed beads to each plate well containing the capture reaction. 3. Set the pipette volume to 40 µl and pipet the mixture up and down at least 5 times to mix thoroughly. 4. Incubate at room temperature for 5 minutes. 5. Place the plate in the magnet and incubate for 2 minutes or until the solution is clear. 6. Carefully remove and discard the supernatant without disturbing the pellet, and add 150 µl of Equalizer Wash Buffer to each reaction. 7. Move the plate side to side in the magnet to wash the beads. Note: If your magnet does not have two positions for shifting the beads, remove the plate from the magnet and gently pipet up and down five times (with the pipettor set to at least half the total volume), then return the plate to the magnet and incubate for 2 minutes or until the solution clears. 8. With the plate still in the magnet, carefully remove and discard the supernatant without disturbing the pellet, and add 150 µl of Equalizer Wash Buffer to each reaction. 9. Repeat the bead wash as described in step With the plate still in the magnet, carefully remove and discard the supernatant. Note: Ensure that as much wash buffer as possible is removed without disturbing the pellet. Elute the equalized library 1. Remove the plate from the magnet and add 100 µl of Equalizer Elution Buffer to each pellet. Seal the plate with MicroAmp adhesive film, vortex thoroughly, and spin down to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times prior to sealing the plate. Note: Spin with enough force to collect droplets, but not pellet beads. If beads are pelleted, vortex again and spin more gently. 2. Elute the library by incubating in a thermal cycler at 32 C for 5 minutes. 3. Place the plate in the magnet and incubate at room temperature for 5 minutes or until the solution is clear. 4. The supernatant contains the equalized library. Proceed to template preparation, or combine or store libraries as described below. Note: The final concentration of each equalized library is ~100 pm. 18 Ion AmpliSeq Exome RDY Library Preparation User Guide

19 Chapter 2 Methods Option 1: Equalize the library 2 (Optional) Combine equalized libraries Store equalized libraries Up to 3 barcoded exome libraries can be combined and run on a single Ion PI chip, depending on the coverage depth desired. Libraries may be stored at 4 8 C for up to 1 month. For longer term storage, store at 20 C. Ion AmpliSeq Exome RDY Library Preparation User Guide 19

20 2 Chapter 2 Methods Option 2: Quantify the unamplified library by qpcr Option 2: Quantify the unamplified library by qpcr Elute the unamplified Ion AmpliSeq library, then determine the concentration by qpcr with the Ion Library Quantitation Kit (Cat. no ). After quantitation, determine the dilution factor that results in a concentration of ~100 pm. Note: The Ion Library Quantitation Kit may also be used to quantify libraries that have been amplified using the procedure described in Amplify the library on page 22. Elute the unamplified library Quantify the library by qpcr and calculate the dilution factor 1. Remove the plate containing the Ion AmpliSeq library from the magnet, and add 50 µl of Low TE to the pellet to disperse the beads. Seal the plate with MicroAmp adhesive film, vortex thoroughly, and spin down to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times prior to sealing the plate. 2. Place the plate in the magnet for at least 2 minutes. The supernatant containts the library. Prepare a 100-fold dilution by removing 5 µl and combining with 495 µl of Nuclease-free Water for quantitation. Determine the concentration of each Ion AmpliSeq library by qpcr with the Ion Library Quantitation Kit using the steps below. Each sample, standard, and negative control should be analyzed in duplicate 20-µL reactions. Unamplified libraries typically have yields of pm. 1. Prepare three 10-fold serial dilutions of the E. coli DH10B Ion Control Library (~68 pm; from the Ion Library Quantitation Kit) at 6.8 pm, 0.68 pm, and pm. Mark these as standards and use these concentrations in the qpcr instrument software. 2. Prepare reaction mixtures. For each sample, control, and standard, combine 20 µl of 2X TaqMan MasterMix and 2 µl of 20X Ion TaqMan Assay and mix thoroughly. Dispense 11-µL aliquots into the wells of a PCR plate. 3. Add 9 µl of the diluted (1:100) Ion AmpliSeq library or 9 µl of each control dilution to each well (two wells per sample as noted before), for a total reaction volume of 20 µl. 4. Program your real-time instrument as follows: Enter the concentrations of the control library standards. Use ROX Reference Dye as the passive reference dye. Select a reaction volume of 20 µl. Select FAM dye/mgb as the TaqMan probe reporter/quencher. The Ion Library TaqMan qpcr Mix can be used on a variety of instruments, as listed below. The fast cycling program was developed using the StepOnePlus System in Fast mode. 20 Ion AmpliSeq Exome RDY Library Preparation User Guide

21 Chapter 2 Methods Option 2: Quantify the unamplified library by qpcr 2 Real-Time PCR System Stage Temp Time 7900 HT System 7900 HT Fast System (Fast 96- Well, Standard 96-Well, or 384- Well Block Modules) ViiA 7 System StepOne System StepOnePlus System 7500 Fast System 7500 System Hold (optional) 50 C 2 min Hold 95 C 20 sec Cycle (40 cycles) 95 C 1 sec 60 C 20 sec Hold (optional) 50 C 2 min Hold 95 C 20 sec Cycle (40 cycles) 95 C 3 sec 60 C 30 sec 5. Following qpcr, calculate the average concentration of the undiluted Ion AmpliSeq library by multiplying the concentration determined with qpcr by Based on the calculated library concentration, determine the dilution that results in a concentration of ~100 pm. For example: The undiluted library concentration is 300 pm. The dilution factor is 300 pm/100 pm = 3. Therefore, 10 µl of library mixed with 20 µl of Low TE (1:3 dilution) yields approximately 100 pm. 7. Dilute library to ~100 pm as described and proceed to combining libraries or template preparation. (Optional) Combine amplicon libraries Store libraries Up to 3 barcoded exome libraries can be combined and run on a single Ion PI chip, depending on the coverage depth desired. Libraries may be stored at 4 8 C for up to 1 month. For longer term storage, store at 20 C. Ion AmpliSeq Exome RDY Library Preparation User Guide 21

22 2 Chapter 2 Methods Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument Ion AmpliSeq libraries must be amplified prior to quantitation to enrich amplifiable material and obtain sufficient material for accurate quantitation. Amplify the library using 1X Library Amp Mix, then purify each library. Quantify the library using the Qubit 2.0 Fluorometer or the Agilent 2100 Bioanalyzer instrument. Note: The Ion Library Quantitation Kit may also be used to quantify libraries that have been amplified using the procedure described in Amplify the library on page 22. After quantitation, determine the dilution factor that results in a concentration of ~100 pm (22 ng/ml for the Qubit 2.0 Fluorometer). Amplify the library 1. Remove the plate containing the Ion AmpliSeq library from the magnet and add 50 µl of 1X Library Amp Mix and 2 µl of 25X Library Amp Primers to each bead pellet. The Library Amp Mix and primers may be combined before addition. 2. Seal the plate with MicroAmp adhesive film, vortex thoroughly, and spin down to collect droplets. Alternatively, mix by pipetting at least half the total volume up and down at least 5 times prior to sealing the plate. Note: Library amplification takes place in the presence of the AMPure XP beads. 3. Place a MicroAmp Compression Pad on the plate, load in the thermal cycler, and run the following program. Stage Temperature Time Hold 98 C 2 min 5 cycles 98 C 15 sec 64 C 1 min Hold 10 C Hold 4. Proceed to Purify the amplified library below. STOPPING POINT Samples may be stored at 20 C. 22 Ion AmpliSeq Exome RDY Library Preparation User Guide

23 Chapter 2 Methods Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument 2 Purify the amplified library Perform a two-round purification process with Agencourt AMPure XP Reagent. First round at 0.5X bead-to-sample-volume ratio: High molecular-weight DNA is bound to beads, while amplicons and primers remain in solution. Save the supernatant. Second round at 1.2X bead-to-original-sample-volume ratio: Amplicons are bound to beads, and primers remain in solution. Save the bead pellet, and elute the amplicons from the beads. IMPORTANT! Bring AMPure XP reagent to room temperature and vortex thoroughly to disperse the beads before use. Pipet solution slowly. Use freshly prepared 70% ethanol for the next steps. Combine 230 µl of ethanol with 100 µl of nuclease-free water per sample. Do NOT substitute a Dynabeads -based purification reagent for the Agencourt AMPure XP reagent. First-round purification 1. Add 25 µl (0.5X sample volume) of Agencourt AMPure XP Reagent to each plate well containing ~50 µl of sample. Pipet up and down 5 times to thoroughly mix the bead suspension with the DNA, and seal the plate with MicroAmp adhesive film. 2. Incubate the mixture for 5 minutes at room temperature. 3. Place the plate in a magnet such as the DynaMag -96 Side Magnet for at least 5 minutes or until the solution is completely clear. 4. Carefully transfer the supernatant from each well to a single well of a new 96-well PCR plate, without disturbing the pellet. IMPORTANT! The supernatant contains the final library. Do not discard! Second-round purification 1. To the supernatant from step 4 above, add 60 µl (1.2X original sample volume) of Agencourt AMPure XP Reagent. Pipet up and down 5 times to thoroughly mix the bead suspension with the DNA, and seal the plate with MicroAmp adhesive film. 2. Incubate the mixture for 5 minutes at room temperature. 3. Place the plate in the magnet for 3 minutes or until the solution is clear. Carefully remove and discard the supernatant without disturbing the pellet. IMPORTANT! The amplicons are bound to the beads. Save the bead pellet. Ion AmpliSeq Exome RDY Library Preparation User Guide 23

24 2 Chapter 2 Methods Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument 4. Add 150 µl of freshly prepared 70% ethanol to each well and move the plate side to side in the magnet to wash the beads. Remove and discard the supernatant without disturbing the pellet. Note: If your magnet does not have two positions for shifting the beads, remove the plate from the magnet and gently pipet up and down five times (with the pipettor set at 100 µl). Return the plate to the magnet and incubate for 2 minutes or until the solution clears. 5. Repeat step 4 for a second wash. 6. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in the magnet, air-dry the beads at room temperature for 5 minutes. Do not overdry. 7. Remove the plate from the magnet and add 50 µl of Low TE to the pellet to disperse the beads. Seal the plate with MicroAmp adhesive film, vortex thoroughly, and spin down to collect droplets. Alternatively, mix by setting a pipettor to 40 µl and pipet the mixture up and down at least 5 times prior to sealing the plate. IMPORTANT! The supernatant contains the desired amplicons. Do not discard! 8. Place the plate in the magnet for at least 2 minutes and analyze an aliquot of the supernatant as described in: Qubit 2.0 Fluorometer: Quantify the library and calculate the dilution factor or Agilent Bioanalyzer : Quantify the library and calculate the dilution factor. 24 Ion AmpliSeq Exome RDY Library Preparation User Guide

25 Chapter 2 Methods Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument 2 Qubit 2.0 Fluorometer: Quantify the library and calculate the dilution factor Analyze 10 µl of each amplified library using the Qubit 2.0 Fluorometer (Cat. no. Q32866) and the Qubit dsdna HS Assay Kit. Amplified libraries typically have concentrations of ng/ml. For more information, see the Qubit dsdna HS Assay Kits User Guide. 1. Determine the amplified library concentration: a. Make a 1:200 working dilution of Qubit dsdna HS reagent using the Qubit dsdna HS Buffer. b. Combine 10 µl of the amplified Ion AmpliSeq library with 190 µl of dye reagent, mix well, and incubate for at least 2 minutes. c. Prepare each Qubit standard as directed in the user guide. d. Measure the concentration on the Qubit 2.0 Fluorometer. e. Calculate the concentration of the undiluted library by multiplying by 20. This can be calculated automatically using the Calculate Stock Concentration button and inputting 10 µl as the sample volume. 2. Based on the calculated library concentration, determine the dilution that results in a concentration of ~100 pm (22 ng/ml). For example: The library concentration is 660 ng/ml. The dilution factor is 660 ng/ml divided by 22 ng/ml = 30. Therefore, 10 µl of library mixed with 290 µl of Low TE (1:30 dilution) yields approximately 22 ng/ml (~100 pm). 3. Dilute library to ~100 pm as described and proceed to combining libraries or template preparation. Agilent Bioanalyzer : Quantify the library and calculate the dilution factor Analyze 1 µl of amplified library on the Agilent Bioanalyzer instrument with the Agilent High Sensitivity DNA Kit (Cat. no ). Amplicon libraries should have multiple peaks in the bp size range. Amplified libraries typically have concentrations of 1,000 10,000 pm. If the library concentration is over 20,000 pm, dilute the library 1:10 and repeat the quantitation to obtain a more accurate measurement. 1. Determine the molar concentration of the amplified library using the Bioanalyzer software. Ensure that the upper and lower marker peaks are identified and assigned correctly. Follow the manufacturer s instructions to perform a region analysis (smear analysis). Briefly: a. Select the Data icon in the Contexts panel, and view the electropherogram of the sample to be quantified. b. Select the Region Table tab below, and create a region spanning the desired amplicon peaks. Correct the baseline if needed. c. The molarity is automatically calculated and displayed in the table in pmol/l (pm). 2. Based on the calculated library concentration, determine the dilution that results in a concentration of ~100 pm. Ion AmpliSeq Exome RDY Library Preparation User Guide 25

26 2 Chapter 2 Methods Option 3: Quantify the amplified library with the Qubit 2.0 Fluorometer or Agilent 2100 Bioanalyzer instrument For example: The library concentration is 3000 pm. The dilution factor is 3000 pm/100 pm = 30. Therefore, 10 µl of library mixed with 290 µl of Low TE (1:30 dilution) yields approximately 100 pm. 3. Dilute library to ~100 pm as described and proceed to combining libraries or template preparation. (Optional) Combine amplicon libraries Store libraries Up to 3 barcoded exome libraries can be combined and run on a single Ion PI chip, depending on the coverage depth desired. Libraries may be stored at 4 8 C for up to 1 month. For longer term storage, store at 20 C. 26 Ion AmpliSeq Exome RDY Library Preparation User Guide

27 3 Combining Ion AmpliSeq libraries Ion Chip capacities for Ion AmpliSeq libraries Up to 3 barcoded exome libraries can be combined and run on a single Ion PI Chip, depending on the coverage and depth desired. The number of exome libraries that can be accommodated in a single sequencing run depends on the size of the chip, the ability to reliably quantify and combine barcoded libraries, and the coverage required. As the number of libraries on a single chip increases, the average coverage depth decreases. This relationship is shown in the following table. The numbers below presume high-accuracy quantitation, and should serve as a guide for approximate capacities. We suggest determining real limits empirically. Multiple chips may be run to increase coverage depth. Run data may be combined by using the CombineAlignments plugin. For detailed instructions on running the CombineAlignments plugin, see Running the Installed Plugins in the Torrent Browser Analysis Report Guide. No. of exomes per Ion PI chip Approximate average read depth * 1 250X 2 125X 3 83X * Actual read depth per sample may vary as a result of barcode balance and chip loading. Ion AmpliSeq Exome RDY Library Preparation User Guide 27

28 A Tips and Troubleshooting Tips and modifications to the standard workflow Tips Plate seals can be firmly applied using the applicator in the MicroAmp Optical Adhesive Film Kit. Plate seals can be removed with much less effort when hot. Try removing seals right after taking the plate out of thermal cycler. Combine and dilute barcode adapters in large batches and aliquot into 96-well plates. If library yield is below 100 pm, libraries may still be sequenced by using a proportionally larger volume into a combined library or into template preparation. Modifications to the standard workflow The following modifications to the standard protocol are designed to allow advanced users to successfully modify and customize the standard Ion AmpliSeq protocol. These modifications are unsupported and in some cases may decrease performance. Limited Samples DNA from high quality FFPE tissue may be used with Ion AmpliSeq Exome. Uniformity and representation of longer amplicons may decrease. When using the Qubit 2.0 or Bioanalyzer instrument, amplified libraries with undetectable product may still be quantified with qpcr ( Option 2: Quantify the unamplified library by qpcr on page 23). Shortcuts When using the Bioanalyzer instrument for quantitation (but not the Qubit 2.0 Fluorometer), a single round of purification at 1.7X volume (85 µl) may be substituted for the two-round purification following library amplification ( Purify the amplified library on page 26). High molecular weight material will not interfere with sequencing, but be sure the markers are assigned correctly. When using qpcr quantitation, careful removal of ethanol after the final wash eliminates the need for drying AMPure XP beads. However, residual ethanol may inhibit the library amplification reaction. 28 Ion AmpliSeq Exome RDY Library Preparation User Guide

29 Appendix A Tips and Troubleshooting Troubleshooting A Troubleshooting Library Yield and Quantitation If troubleshooting does not resolve the problem, contact Technical Support. Observation Possible cause Recommended action Low library yield with the Ion Equalizer Kit Unwashed beads Residual salt after wash Be sure to wash Equalizer Beads prior to use Carefully remove all wash solution prior to elution Low library yield general Less than 100 ng of input DNA Add more DNA or increase target amplification cycles to 11 or 12 ( Amplify targets on page 16) Inefficient PCR, digestion, or ligation Library discarded during purification of the amplified library Over-drying of AMPure XP beads Residual ethanol drops inhibiting library amplification Mis-quantitation of input DNA AMPure beads inhibiting library amplification Ensure proper dispensing and mixing of viscous components at each step Be sure to save the supernatant during first-round purification, and save the bead pellet during the second round ( Purify the amplified library on page 26) Do not dry the AMPure XP beads more than 5 minutes Carefully remove all drops, using an additional spin and removal step, if necessary Requantify input DNA using the TaqMan RNase P Detection Reagents Kit Transfer library off of beads prior to amplification High library yield Mis-quantitation of input DNA Quantify input DNA using the TaqMan RNase P Detection Reagents Kit High library yield on the Bioanalyzer instrument High molecular weight material on the Bioanalyzer instrument (Figure 2) or High library yield on the Qubit Fluorometer More than 100 ng of input DNA Marker mis-assignment (Figure 1) High molecular weight DNA was not removed during purification of the amplified library (does not interfere with sequencing) Add less DNA or decrease target amplification cycles to 9 ( Amplify targets on page 16) Ensure that markers are assigned correctly Carefully remove less supernatant in the firstround (0.5X) purification ( First-round purification on page 26) and be sure not to disturb bead pellet Increase AMPure XP Reagent volume from 25 to 35 µl (0.5X to 0.7X) in the first-round purification ( First-round purification on page 26) Ion AmpliSeq Exome RDY Library Preparation User Guide 29

30 A Appendix A Tips and Troubleshooting Troubleshooting Bias in amplicon representation Observation Possible cause Recommended action Loss of short amplicons Poor purification Vortex AMPure XP Reagent thoroughly before use, and be sure to dispense the full volume In unamplified library purification ( Purify the unamplified library on page 19), increase AMPure XP Reagent volume from 80 to 96 µl (1X to 1.2X) In amplified library purification ( Purify the amplified library on page 26), increase AMPure XP Reagent volume in second round from 60 to 70 µl (1.2X to 1.4X) Denaturation of digested amplicon Verify use of the 60 C/20 minute temperature incubation during the primer digestion step ( Partially digest primer sequences on page 18) Loss of long amplicons Low quality DNA FFPE DNA is not recommended for Ion AmpliSeq Exome RDY Loss of AT-rich amplicons (see Figure 4) Loss of GC-rich amplicons (see Figure 5) Uneven pool representation (see Figure 6) Too few nucleotide flows Denaturation of digested amplicon Unknown Inadequate denaturation Inefficient library amplification Inadequate amplification Unknown Inaccurate pipetting Inaccurate PCR product combination Use at least 500 flows to sequence through exome amplicons Verify use of the 60 C/20 minute temperature incubation during the primer digestion step Amplicons with >80% AT often exhibit low representation Use a calibrated thermal cycler Do not amplify the library (not required for qpcr quantitation) Increase the anneal/extend temperature of the target amplification reaction from 60 C to 62 C for the first two cycles of the target amplification reaction and increase cycle number if necessary Amplicons with >80% GC often exhibit low representation Thoroughly mix target amplification master mix and dispense equal volumes into each well Carefully combine all target amplification products without changing tips. Centrifuge plate and transfer residual droplets, if needed. 30 Ion AmpliSeq Exome RDY Library Preparation User Guide

31 Appendix A Tips and Troubleshooting Troubleshooting A Other Observation Possible cause Recommended action Adapter dimers on the Bioanalyzer instrument at bp (see Figure 3,) or Adapter dimers during sequencing Lower-than-expected number of on-target reads Uneven barcode representation High polyclonal ISPs (>40%) High low quality ISPs (>15%) Inefficient purification Adapter dimer formation Adapter concentration too high In unamplified library purification ( Purify the unamplified library on page 19), decrease AMPure XP Reagent volume from 80 to 60 µl (1X to 0.75X) In amplified library purification ( Purify the amplified library on page 26), decrease AMPure XP Reagent volume in second round from 60 to 50µL (1.2X to 1X) Do not combine Adapters, DNA ligase, and Switch solution prior to addition Use a 65 C temperature incubation instead of 60 C during the primer digestion step ( Partially digest primer sequences on page 18). Ensure that barcode adapters are diluted properly Unknown Increase the number of target amplification cycles by 10 to 12, and/or increase the anneal/extend temperature of the target amplification reaction from 60 C to 62 C for the first two cycles of the target amplification reaction Inaccurate library quantitation Inaccurate library combination Overseeding of library Library mis-quantitation Other Underseeding of library Library mis-quantitation Other Use the Ion Library Quantitation Kit for the most specific and accurate library quantitation Dilute libraries to 100 pm, then combine equal volumes Decrease amount of library added to the template preparation reaction by 50% Ensure that library was quantified correctly Check the appropriate template preparation user guide for more information. Double the volume of library used in template prep Use a fresh dilution of library prepared in a low-bind tube Ensure that library was quantified correctly Check the appropriate template preparation user guide for more information. Ion AmpliSeq Exome RDY Library Preparation User Guide 31

32 A Appendix A Tips and Troubleshooting Troubleshooting Figure 1: Marker mis-assignment. Marker mis-assignment on the Agilent 2100 Bioanalyzer instrument. When the upper marker is assigned to the incorrect peak, library yield may be overestimated by more than 10-fold. Figure 2: High molecular weight material in an Ion AmpliSeq Exome library. High molecular weight material on Bioanalyzer instrument. DNA outside the region window will not interfere with template preparation or sequencing, but may lead to an overestimation of library concentration when using the Qubit 2.0 Fluorometer for library quantitation. 32 Ion AmpliSeq Exome RDY Library Preparation User Guide

33 Appendix A Tips and Troubleshooting Troubleshooting A Figure 3: Adapter dimers. Standard adapters produce peaks at ~43 and ~53 bp, while adapter dimers run at ~90 bp. Barcode adapters run at ~53 bp, and barcode adapter dimers run at ~105 bp. Figure 4: Example of loss of AT-rich amplicons. Within the Coverage Analysis Plugin, amplicon representation is plotted by GC content for the Ion AmpliSeq Exome Panel. Amplicons with 23-50% GC content show an excess failure rate (less than 20% of the mean read depth). Ion AmpliSeq Exome RDY Library Preparation User Guide 33

34 A Appendix A Tips and Troubleshooting Troubleshooting Figure 5: Example of loss of GC-rich amplicons. Within the Coverage Analysis Plugin, amplicon representation is plotted by GC content for the Ion AmpliSeq Exome Panel. Amplicons with 50-80% GC content show an excess failure rate (less than 20% of the mean read depth). Figure 6: Example of pool imbalance. Within the Coverage Analysis Plugin, mean read depth per primer pool is plotted for Ion AmpliSeq Exome. In this example, pool 1 has approximately half the reads of other pools. 34 Ion AmpliSeq Exome RDY Library Preparation User Guide

35 B Supplemental information Data analysis Visit the Ion Community at and select Products Systems and Software Torrent Suite to access the latest user guides and information for Torrent Suite software. Torrent Suite is required for sequence analysis of libraries prepared with Ion AmpliSeq Primer Pools. The software includes the Torrent Variant Caller Plugin, which can be used to call single nucleotide polymorphism (SNP) and insertion/ deletion (InDel) variants within the genomic regions covered by the Ion AmpliSeq Primer Pools. To enable variant calling: Install the hg19 human genome reference on your Torrent Server, if it is not already installed. Upload the BED files for the Ion AmpliSeq Primer Pool target regions to your Torrent Server, or use the analysis plan available at AmpliSeq.com. Install the hg19 reference on your Torrent Server Brief instructions for installing the hg19 reference on your Torrent Server are provided below. Detailed instructions are provided under Adding a Reference Sequence in the Torrent Browser User Interface Guide. 1. Download the FASTA file from hg19.zip. 2. In the Torrent Browser, click the Settings button on the right side of the screen and select References. 3. Click on Add Reference Sequence and enter the information in the fields. In the Short Name field, the short form of the genome name must be entered as hg19. The genome index creation will take a few hours. Ion AmpliSeq Exome RDY Library Preparation User Guide 35

36 B Appendix B Supplemental information Data analysis Import the BED files into your Torrent Server Coverage Analysis Plugin Browser Extensible Data (BED) files supply chromosome regions and restrict analysis to the regions in the designated Ion AmpliSeq panel. These two types of BED files are frequently used: Designed BED file - Specifies the amplified regions that are used with targeted sequencing. Hotspot BED file - Specifies regions of known mutations, for example from COSMIC or dbsnp databases or from customer-defined regions. A hotspot file is optional. Positions in a hotspot file will always be present in the VCF file and in the variant table in Torrent Suite software and Ion Reporter. They may have a variant present, or they may be reference, or no-call. All alleles present at this position, with the number of reads of each allele, will always be shown at Hotspot positions. In addition, Hotspots can be called with different thresholds (for example, lower allele frequency). To import and download the BED files you require, follow the steps listed in Create a Template with AmpliSeq.com Import in the Torrent Browser User Interface Guide. The Coverage Analysis Plugin provides statistics and graphs describing the level of sequence coverage produced for targeted genomic regions. You can run the Coverage Analysis Plugin automatically or manually. Option 1: Include the Coverage Analysis Plugin in a run plan To run the Coverage Analysis Plugin automatically, select the Coverage Analysis plugin during template setup. Refer to the Plan Tab and Templates sections of the Torrent Browser User Interface Guide for information about how to set up a template and create a planned run. Option 2: Manually launch the Coverage Analysis Plugin To run the Coverage Analysis Plugin manually: 1. Open your run report on the Torrent Browser. 2. Scroll to the Plugin Summary area. Click Select plugins to run. The Select a plugin popup appears: 3. Select CoverageAnalysis to launch the Coverage Analysis Plugin interface. 36 Ion AmpliSeq Exome RDY Library Preparation User Guide

37 Appendix B Supplemental information Data analysis B 4. Select a Library Type: either Ion AmpliSeq or Ion AmpliSeq Exome. 5. Select your designed BED file in the Targeted Regions pull-down menu. 6. Check the boxes for the remaining options if desired: Barcode-specific Targets - If you are running multiple Ion AmpliSeq libraries created from different primer pools, you can select barcode-specific target BED files to analyze against. Use Only Uniquely Mapped Reads - If you would like the plugin to examine only reads that map preferably to a single location, check this box. 7. When you are satisfied with your selections, click Submit. Coverage Analysis Report The run report will include basic run metrics, but more detail can be found by clicking on the coverageanalysis.html link, including options to download a barcode summary report, and an amplicon coverage matrix. More detailed information can be obtained for each barcoded library by clicking the Barcode ID. Ion AmpliSeq Exome RDY Library Preparation User Guide 37

38 B Appendix B Supplemental information Data analysis At the top of the Coverage Analysis Report you will find a table that shows amplicon statistics on the left side and base statistics on the right side. All metrics are defined with flyover explanations. The plugin run options may be reviewed as flyover help on the report title Coverage Analysis Report. The Representation Plots (which are revealed by clicking on the triangle in the blue bar) allow you to visualize amplicon representation by GC content and length. If the designed BED file defines multiple primer pools, a mean target depth per primer pool representation plot is also available. In the example below, pool 1 is under represented: The Amplicon Coverage Chart shows amplicons binned by representation, low to high, with a variety of overlays. You can zoom into this graph to see how many amplicons are in each bin, and click on a bar to get more information about that group of amplicons. 38 Ion AmpliSeq Exome RDY Library Preparation User Guide

39 Appendix B Supplemental information Data analysis B By clicking on the magnifying glass in the upper right corner of the Amplicon Coverage Chart, you can change and filter what is shown on the chart by number of reads, chromosome, or gene. The Reference Coverage Chart shows the strand-specific coverage in red and green for each amplicon. The example below shows the coverage for each exon of the TP53 gene: Finally, at the bottom of the Coverage Analysis Report you will find links to download the data in this report as well as the BAM and BAI files required for IGV. Ion AmpliSeq Exome RDY Library Preparation User Guide 39

40 B Appendix B Supplemental information Data analysis Torrent Variant Caller Plugin For detailed instructions on running the Torrent Variant Caller, refer to "Running the Torrent Variant Caller Plugin" in the Torrent Browser Analysis Report Guide. The Torrent Variant Caller (TVC) Plugin calls single-nucleotide polymorphisms (SNPs), multinucleotide polymorphisms (MNPs), insertions, and deletions in a sample across a reference or within a targeted subset of that reference. This plugin provides optimized pre-set parameters for many experiment types, but is also very customizable. After you find a parameter combination that works well on your data and that has the balance of specificity and sensitivity that you want, you can save that parameter set and reuse it over and over in your research. This is supported on both manual launches of the plugin and in automatic launches through the run plan template wizard. Note: The TVC plugin run uses the same target regions file and hotspots file as the main Torrent Suite Software analysis (if those files are present in the main analysis). Through the run plan wizard there is no capability in the TVC configuration to change the target regions file or hotspots file. You can use a different target regions file and hotspots file with a manual TVC launch from a completed run report. Parameters TVC provides several ways of handling its parameter options: You can import parameter settings that are optimized for fixed panels and community panels from AmpliSeq.com (click on Panel Files TVC settings are.json files). You can select one of TVC default pre-set parameter groups or customize them in the TVC UI. The parameters used in a TVC run can also be downloaded and modified, or reused in future TVC runs. TVC's default parameters setting groups are organized according to these attributes: Variant frequency - Somatic settings are optimized to detect low frequency variants. Germ-line settings are optimized for high frequency settings. Sequencing instrument - The Ion PGM or the Ion Proton sequencer. Parameter defaults are different for Ion Proton data than for Ion PGM data. Stringency - High stringency settings are optimized to minimize false positives. Low stringency settings minimize false negatives. Run the Torrent Variant Caller Plugin There are two ways to run the TVC Plugin: Automatically, by preconfiguring the plugin during the run planning or Manually, allowing you to run the plugin at any time from a completed run report. 40 Ion AmpliSeq Exome RDY Library Preparation User Guide

41 Appendix B Supplemental information Data analysis B 1. When you select the Plugins chevron in the template or Run Plan Wizard and enable the variantcaller checkbox, a Configure link appears: 2. Click the Configure link to open the variantcaller configuration popup: 3. Select one of the pre-defined parameter settings or upload your own custom settings file (see Ampliseq.com for the best settings for the panel you are using). Torrent Variant Caller Plugin output The TVC plugin output includes the following reports and sections: Run report plugin summary Variant Caller Report Variant Caller Report summary section Variant Caller Report variant calls table Ion AmpliSeq Exome RDY Library Preparation User Guide 41

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