Prepare Ion AmpliSeq Libraries using the Tecan Freedom EVO NGS Workstation

Transcription

1 USR ULLTIN Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation Publication Number MN Revision.0 Protocol information... 2 Ion kits used in this protocol... 3 Input N requirements... 7 Input RN requirements... 7 Using the Tecan reedom VO NS Workstation... 7 Prepare and amplify cn targets... 9 mplify N targets Thermal cycling conditions Partially digest primer sequences Ligate adapters to the amplicons and purify Option 1: qualize the library Option 2: Purify and quantify the unamplified library by qpr Option 3: Quantify the amplified library with Qubit 2.0 luorometer or gilent 2100 ioanalyzer Quantify the library using the Qubit 2.0 luorometer Quantify the library using gilent 2100 ioanalyzer Instrument (Optional) ombine amplicon libraries Store libraries ocumentation and support or Research Use Only. Not for use in diagnostic procedures.

2 Protocol information Protocol information Revision history Revision ate escription.0 2 ebruary 2014 dded RN workflow dded "ill volume" columns to worktable set up tables.0 10 October 2014 orrected callouts in images and corresponding tables in the following sections:.0 29 ugust 2014 irst release Purify, elute, and amplify the library on page 22 dd qualizer beads and wash on page 25 mplify the library on page 30 escription This user bulletin describes how to prepare Ion mpliseq libraries using the Tecan reedom VO NS Workstation. The workflow for library preparation is similar to that of the Ion mpliseq library kits, with additional steps to set up the Tecan reedom VO NS Workstation, import and run the scripts. or more information, refer to the Ion mpliseq N and RN Library Preparation uide (Pub. no. MN ). Procedure overview mplify target regions from N and treat the resulting amplicons with upa Reagent to partially digest the primers and phosphorylate the amplicons. Next, ligate the amplicons to adapters with barcodes and purify them. Normalize or quantify libraries and combine them (optional) prior to template preparation and sequencing. Note: The automated method described here is not compatible with Ion mpliseq xome Kits. The following kits are used in this automation protocol: Ion mpliseq Library Kit LV or 384LV (at. no or ): One or more kits are required for preparing libraries using the automated platform to accommodate for higher dead volume requirements. ach kit contains reagents for the rapid preparation of either 96 or 384 libraries containing 12 24,576 amplicons per reaction. ach kit uses a 96-well plate-based protocol for easy sample handling and tracking, and for compatibility with automation and high-throughput laboratories. Ion Library qualizer Kit (at. no ): This kit provides an optional streamlined method for normalizing library concentration without the need for quantitation. Ion Xpress arcode dapters (at. no ): One or more kits are required for preparing barcoded libraries. ach kit includes reagents sufficient for preparing up to 40 Ion mpliseq libraries per barcode (40 16 libraries). 2 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

3 Ion kits used in this protocol Ion kits used in this protocol Ion mpliseq Library Kit LV or 384LV The Ion mpliseq Library Kit LV or 384LV (at. no or ) provides reagents for preparing 96 and 384 libraries, respectively. If preparing the maximum number of libraries per kit, multiple kits may be required to accommodate for higher dead volume requirements associated with the automated platform. Ion mpliseq Library Kit LV or 384LV (at. no or ) Number of tubes omponent ap color at. no at. no Volume per tube Storage 5X Ion mpliseq ii Mix Red 1 tube 4 tubes 384 µl 30º to 10º upa Reagent rown 1 tube 4 tubes 192 µl Switch Solution Yellow 1 tube 4 tubes 384 µl N Ligase lue 1 tube 4 tubes 192 µl Ion mpliseq dapters reen 1 tube 4 tubes 192 µl Platinum PR SuperMix ii lack 3 tubes 12 tubes 1.6 ml Library mplification Primer Mix White 1 tube 4 tubes 192 µl Low T lear 1 tube 4 tubes 12 ml Room temp (15 to 30 ) Ion Library qualizer Kit The Ion Library qualizer Kit contains reagents for 96 reactions. Ion Library qualizer Kit (at. no ) omponent ap color Quantity Volume per tube Storage Ion Library qualizer Primers Pink 1 tube 200 µl 2º to 8º Ion Library qualizer apture Purple 1 tube 1 ml Ion Library qualizer lution uffer Switch Solution lear 1 bottle 10 ml Ion Library qualizer eads Orange 1 tube 300 µl Ion Library qualizer Wash uffer lear 1 bottle 35 ml Room temp (15º to 30º) Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 3

4 Ion kits used in this protocol Ion Xpress arcode dapters One or more Ion Xpress dapters Kits are required for preparing barcoded libraries. ach kit includes reagents sufficient for preparing up to 40 Ion mpliseq libraries per barcode (40 16 libraries). Substitute Ion Xpress dapters for standard Ion mpliseq dapters as described in this user guide. Ion Xpress arcode dapters (Various at. nos. ach kit includes 16 individually numbered barcodes) omponent ap color Quantity Volume per tube Storage Ion Xpress P1 dapter Violet 1 tube 320 µl 30º to 10º Ion Xpress arcode X White 16 tubes (1 per barcode) 20 µl each 4 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

5 Ion kits used in this protocol Required materials and equipment Unless otherwise specified, all materials are available from Life Technologies ( MLS: isher Scientific ( or major laboratory supplier. escription Supplier atalog no. Quantity Tecan reedom VO NS Workstation Tecan See NS 1 Tecan Pure Lia isposable Tips, iltered, 50, 200, and 1000 µl Tecan , , and isposable Reagent Troughs, 25 ml and 100 ml Tecan and ppendorf eepwell Plates, 500 µl ppendorf Micromp nduraplate Optical 96-well Reaction Plates with arcode Life Technologies Micromp Splash-ree 96-well ase Life Technologies One of the following: enemp PR System 9700 or ual 96- well Thermal ycler 2720 Thermal ycler Veriti 96-well Thermal ycler Prolex 96-Well PR System Life Technologies See web product pages 1 96-well plate centrifuge MLS Various 1 (RN only) SuperScript VILO cn Synthesis Kit Life Technologies reactions Micromp dhesive ilm Life Technologies Micromp ompression Pad Life Technologies set gencourt MPure XP Kit eckman oulter or ynamag Side Magnet, or other plate magnet Life Technologies Nuclease-ree Water Life Technologies M ml bsolute ethanol MLS N/ ~15 ml (Optional) Recoverll Total Nucleic cid Isolation Kit for P (Optional) MagMX P Total Nucleic cid Isolation Kit Life Technologies M preps Life Technologies preps (Optional) PureLink enomic N Mini Kit Life Technologies K preps (Recommended for N quantitation) TaqMan RNase P etection Reagents Kit Life Technologies Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 5

6 Ion kits used in this protocol escription Supplier atalog no. Quantity (Optional for library quantitation) If you are not using the Ion Library qualizer Kit (at. no ) for library normalization, select one of the following: Qubit 2.0 luorometer and Qubit dsn S ssay Kit gilent 2100 ioanalyzer and gilent igh Sensitivity N Kit Life Technologies Q32866, Q32851/ Q32854 gilent 2939, Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

7 Input N requirements Input N requirements N isolation and quantitation mount of N needed See Required materials and equipment on page 5 for recommended kits for isolating N. We recommend the TaqMan RNase P etection Reagents Kit (at. no ) for quantitating amplifiable human genomic N (see ioncommunity.lifetechnologies.com/docs/o-7431). The Qubit dsn S ssay Kit (at. no. Q32851 or Q32854) may also be used. such as densitometry (e.g., Nanorop Spectrophotometers) are not recommended, because they do not discriminate between N and RN and thus are extremely sensitive to small fragments of hydrolyzed RN. This can lead to gross overestimation of the concentration of sample N, underseeding of the target amplification reaction, low quality libraries, and low library yields. ach target amplification reaction requires 10 ng of 2 ng/μl genomic N (gn) or N from P (at least 8 μl for each reaction). Note: e sure to use an P-compatible Ion mpliseq panel for N isolated from P tissue. Standard designs with longer amplicons may perform poorly with N from P tissue. Input RN requirements mount needed and quantitation In general, the library yield from high quality RN is greater than from degraded samples. Library yield is not indicative of sequencing performance. See Required materials and equipment on page 5 for kits recommended for isolating RN. ach reverse transcription reaction requires 10 ng of Nase-treated RN (³ 1.4 ng/μl), prepared from normal or P tissue. We recommend the Qubit RN S ssay Kit (at. no. Q32855) for quantitating RN. Using the Tecan reedom VO NS Workstation Import the script file 1. ownload and extract the mpliseq_n_rn.exd.zip from the Ion ommunity ( 2. Open the Tecan xport Import tool 3. Select ile > Load > Importfile. 4. Select "mpliseq_n_rn.exd" and select Open. 5. Select Import ll to import the scripts. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 7

8 Using the Tecan reedom VO NS Workstation Tip handling subroutine within all scripts contains a method for tracking and handling 50-μL and 200-μL tips. You only need to replace tips when all six boxes are empty. lternatively, at the start of a run you can replace all tip boxes and enter a value of "1" when prompted with "New tips boxes?" This will reset the tip count. The 1000-μL tips are handled separately and replaced as necessary No. Tip type Location(s) 200 µl (blue) rid 3, Site 1 rid 22, Sites 1 2 Storage rack positions µl (green) rid 3, Site 2 rid 22, Sites 3 4 Storage rack positions µl (yellow) rid 16, Site 1 8 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

9 Prepare and amplify cn targets Prepare and amplify cn targets Reverse transcribe RN If RN was prepared from P tissue and not previously heat-treated, pre-heat at 80 for 10 minutes, then cool to room temperature. Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: Table 1 Reagent setup summary: Reverse transcribe RN No. Position escription Labware ill volume [1] rid 2, Site 1, Well 1 (Standard Transfer Only) rid 2, Site 1, Well 2 (Standard Transfer Only) rid 10, Site 1 (ast Transfer Only) 10X SuperScript III nzyme Mix 0.5-mL tube 120 µl [2] 5X VILO RT Reaction Mix 0.5-mL tube 240 µl [2] 10X SuperScript III nzyme Mix 5X VILO RT Reaction Mix Micromp nduraplate reaction plate rid 10, Site 2 RT plate Micromp nduraplate reaction plate rid 10, Site 3 RN plate Micromp nduraplate [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] ombine two tubes from SuperScript VILO cn Synthesis Kit. reaction plate 10X SuperScript III nzyme Mix : 20 µl/well in column 2 5X VILO RT Reaction Mix: 40 µl/well in column 4 mpty 8 µl/well (1.4 ng/µl) 2. dd the following reagents according to the transfer mode selected: Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 9

10 Prepare and amplify cn targets Transfer Mode Reagent Setup Standard 1. Place the tube containing 10X SuperScript III nzyme Mix in position 1 of the chilled metal cooling block at rid 2, Site 1 (). 2. Place the tube containing 5X VILO RT Reaction Mix in position 2 of the chilled metal cooling block at rid 2, Site 1 (). ast 1. liquot 10X SuperScript III nzyme Mix into all wells of column 2 and 5X VILO RT Reaction Mix into all wells of column 4 in a Micromp nduraplate. 2. Place the plate on the incubator at rid 10, Site 1 (). 3. Place a new Micromp nduraplate reaction plate on the incubator at rid 10, Site 2 (). 4. Place Micromp nduraplate reaction plate containing RN (1.4 ng/μl) on the incubator at rid 10, Site 3 (). 5. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "RN_RT." 4. nter the number of reactions to be prepared. 5. If you are using ast Mode, enter "1" when prompted "nable ast Transfer?." Otherwise leave the value as "0." 6. lick RUN. The run time is ~45 minutes (Standard) or 20 minutes (ast) for 96 reactions. 7. When the script is complete, seal the plate with a Micromp dhesive ilm. Vortex the plate 3 times for three seconds each at setting 7 10, then centrifuge the plate briefly. 8. Place a Micromp ompression Pad on the plate, load it in the thermal cycler, and run the following program: Temperature Time min 85 5 min 10 old STOPPIN POINT Samples may be stored at 10 overnight (12 16 hours) in the thermal cycler. or longer periods, store at Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

11 Prepare and amplify cn targets mplify cn targets Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: Table 2 Reagent setup summary: mplify cn No. Position escription Labware ill volume [1] rid 1, Site 1 Water 25-mL reservoir 1 ml rid 2, Site 1, Well 1 5X ii Mix (red cap) 0.5-mL tube 500 µl [2] rid 2, Site 1, Well 2 5X RN Primer Panel 1.5-mL tube 500 µl rid 4, Site 3 eepwell plate ppendorf eepwell plate 96/500 rid 10, Site 2 cn plate nduraplate reaction plate mpty rom previous reaction [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] To ensure sufficient volume for 96 reactions, add 50 μl of 5X ii Mix to a full tube from a 96-LV kit to accommodate dead volumes. This tube may be refilled from another tube later to minimize reagent loss. 2. ill a 25-mL trough with Nuclease-ree Water and place it inside a 100-mL trough at rid 1, Site 1 (). 3. Place the tube containing 5X Ion mpliseq ii Master Mix (red cap) in position 1 of the chilled metal cooling block at rid 2, Site 1 (). 4. Place the tube containing 5X Ion mpliseq RN panel in position 2 of the chilled metal cooling block at rid 2, Site 1 (). 5. Place a 500-μL plate onto the shaking incubator at rid 4, Site 3 (). Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 11

12 Prepare and amplify cn targets 6. Place the Micromp nduraplate reaction plate containing the cn from the previous reaction on the incubator at rid 10, Site 2 (). 7. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "RN_mp." 4. nter the number of reactions to be prepared. 5. lick RUN. The run time is ~10 minutes for 96 reactions. 6. When the script is complete, seal the plate with Micromp dhesive ilm and spin down to collect the droplets. 7. Proceed immediately to Thermal cycling conditions on page Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

13 mplify N targets mplify N targets Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: Table 3 Reagent setup summary: mplify N No. Position escription Labware ill volume [1] rid 1, Site 1 Water 25-mL reservoir 1 ml rid 2, Site 1, Well 1 5X ii Mix (red cap) 0.5-mL tube 500 µl [2] rid 2, Site 1, Well 2 Primer Mix (diluted to 2X) [3] 1.5-mL tube 1.2 ml rid 2, Site 1, Well 5 (Optional) 20X Sample I Panel rid 4, Site 3 eepwell plate ppendorf eepwell plate 96/500 rid 10, Site 2 mplification plate (output) 0.5-mL tube 120 µl nduraplate reaction plate mpty mpty rid 10, Site 3 gn nduraplate reaction plate 8 µl/well, 2 ng/µl [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] To ensure sufficient volume for 96 reactions, add 50 μl of 5X ii Mix to a full tube from a 96-LV kit to accommodate dead volumes. This tube may be refilled from another tube later to minimize reagent loss. [3] Single primer pool only. IMPORTNT! The script uses primers at 2X concentration only. If you are using a 5X primer pool, dilute to 2X with Nuclease-ree Water in a 1.5-mL tube prior to use. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 13

14 mplify N targets 2. ill a 25-mL trough with Nuclease-ree Water and place it inside a 100-mL trough at rid 1, Site 1 (). 3. Place the tube containing 5X Ion mpliseq ii Master Mix (red cap) in position 1 of the chilled metal cooling block at rid 2, Site 1 (). 4. Place the tube containing 2X Ion mpliseq Primer Pool in position 2 of the chilled metal cooling block at rid 2, Site 1 (). 5. Optional: Place the tube containing 20X Ion mpliseq Sample I Panel in position 5 of the chilled metal cooling block at rid 2, Site 1 (). Note: If you are using the Sample I Panel with a ustom or Ready-to-use Panel containing multiple primer pools, you only need to add the Sample I Panel to one of the target amplification reactions. 6. Place a 500-μL plate onto the shaking incubator at rid 4, Site 3 (). 7. Place a new Micromp nduraplate reaction plate on the incubator at rid 10, Site 2 (). 8. Place the Micromp nduraplate reaction plate containing the prepared gn (2 ng/μl) on the incubator at rid 10, Site 3 (). 9. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "Target_mp." 4. nter the number of reactions to be prepared. 5. lick RUN. The run time is ~20 min for 96 reactions. 6. When the script is complete, seal the plate with Micromp dhesive ilm and spin down to collect the droplets. 7. Proceed immediately to Thermal cycling conditions on page Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

15 Thermal cycling conditions Thermal cycling conditions Place a Micromp ompression Pad on the plate, load the plate in the thermal cycler, and run the following program to amplify target N regions. Stage Step Temperature Time old ctivate the enzyme 99 2 min ycle; set number according to the following table enature sec nneal and extend 60 4 min/8 min/ 16 min [1] old 10 old [2] [1] 4 minutes for 1536 primer pairs per pool; 8 minutes for 1,537 6,144; 16 minutes for 6,145 24,576. [2] Samples may be held at 4 overnight. Primer pairs per pool (see notes below) Recommended number of amplification cycles Normal N/RN P N/RN gene fusion , ,537 3, ,073 6, ,145 12, ,289 24, Note: Ready-to-use panels: The Ion mpliseq ancer otspot Panel v2 is 207 primer pairs, the omprehensive ancer Panel is ~4,000 primer pairs/pool, and the Inherited isease Panel is ~3,500 primer pairs/pool. Note: ycle numbers can be increased when input material quality or quantity is questionable. The cycle number does not need to be adjusted when using the Ion mpliseq Sample I panel. STOPPIN POINT PR products may be stored at 10 overnight. or longer periods, store at 20. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 15

16 Partially digest primer sequences Partially digest primer sequences Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: : Table 4 Reagent setup summary: Partially digest primers No. Position escription Labware ill volume [1] rid 2, Site 1, Well 1 (Standard Transfer Only) rid 10, Site 1 (ast Transfer Only) upa Reagent (brown cap) 0.5-mL tube 240 µl [2] upa Reagent nduraplate reaction plate rid 10, Site 2 mplified N nduraplate reaction plate [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] Use a full tube(s) from a 96-LV kit to accommodate dead volumes. 40 µl/well in column 1 rom previous reaction 2. dd the following reagents according to the transfer mode selected: Transfer Mode Reagent Setup Standard 1. Place the tube containing upa Reagent (brown cap) in position 1 of the chilled metal cooling block at rid 2, Site 1 (). ast 1. liquot upa Reagent (brown cap) into all wells of column 1 in a Micromp nduraplate reaction plate and place iton the incubator at rid 10, Site 1 (). This plate can be stored at -20 for later use. 16 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

17 Partially digest primer sequences 3. arefully remove the plate seal from the amplified N plate and place it on the incubator at rid 10, site 2 (). 4. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "upa." 4. nter the number of reactions to be prepared. 5. If you are using ast Mode, enter "1" when prompted "nable ast Transfer?." Otherwise leave value as "0." 6. lick RUN. The run time is ~20 minutes (Standard) or 7 minutes (ast) for 96 reactions. 7. When the script is complete, seal the plate with Micromp dhesive ilm. Vortex the plate three times for 3 seconds each at setting 7 10, then centrifuge the plate briefly. 8. Place a Micromp ompression Pad on the plate, load the plate in the thermal cycler, and run the following program: Temperature Time min min min 10 old (for up to 1 hour) Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 17

18 Ligate adapters to the amplicons and purify Ligate adapters to the amplicons and purify arcoded libraries only: ombine and dilute adapters If you are running multiple sample libraries on a single chip, you can assign a unique barcode to each library. or each barcode X chosen, prepare a mix of Ion P1 dapter and Ion Xpress arcode X at a final dilution of 1:4 for each adapter in a Micromp nduraplate 96-well plate. IMPORTNT! When handling barcoded adapters, be careful to avoid crosscontamination by changing gloves frequently and opening one tube at a time. Table 5 xample barcode adapter mix for up to 40 reactions omponent Volume Ion P1 dapter 20 µl Ion Xpress arcode X [1] 20 µl Nuclease-ree Water 40 µl Total 80 µl [1] X = arcode chosen IMPORTNT! arcodes must be arrayed in the following pattern (i.e., barcode number must match position number on the plate): 18 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

19 Ligate adapters to the amplicons and purify Ligate adapters Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: : Table 6 Reagent setup summary: Ligate adapters No. Position escription Labware ill volume [1] rid 2, Site 1, Well 1 (Standard Transfer Only) rid 2, Site 1, Well 2 (Standard Transfer Only) rid 10, Site 1 (ast Transfer Only) Ligase (blue cap) 0.5-mL tube 240 µl [2] Switch solution (yellow cap) Ligase and Switch Solution 0.5-mL tube 480 µl [2] nduraplate reaction plate rid 10, Site 2 igested N nduraplate reaction plate rid 10, Site 3 arcode Plate (5 µm each) [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] Use a full tube(s) from a 96-LV kit to accommodate dead volumes. nduraplate reaction plate Switch Solution: 80 µl/well in column 3 Ligase: 40 µl/well in column 5 rom previous reaction 8 µl/well 2. dd the following reagents according to the transfer mode selected: Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 19

20 Ligate adapters to the amplicons and purify Transfer Mode Reagent Setup Standard 1. Place the tube containing Switch Solution (yellow cap) in position 2 of the chilled metal cooling block at rid 2, Site 1 (). 2. Place the tube containing Ligase in position 1 of the chilled metal cooling block at rid 2, Site 1 (). ast 1. liquot Switch Solution (yellow cap) into all wells of column 3 and Ligase into all wells of column 5 in a Micromp nduraplate reaction plate and place it on the incubator at rid 10, Site 1 (). This plate can be stored at -20 for later use. 3. arefully remove the plate seal from the amplified and partially digested N plate (from Partially digest primer sequences on page 16) and place it on the incubator at rid 10, Site 2 (). 4. Place the arcode Plate with the diluted barcode adapter mix in the appropriate locations on the incubator at rid 10, Site 3 (). 5. Replace the tip boxes as needed. arcode transfer 1. ownload the file "Ion mpliseq dapter Worklist PLT.xlsm" from the Ion ommunity ( 2. Open the file and enable the macros. 3. or each reaction position, assign a barcode by typing a number (1 96) into the cell. Note: The instrument will always prepare libraries from top to bottom and left to right. The position on the plate map refers to the library position, and the number entered into each cell refers to the well number on the diluted barcode source plate. The example below assigns various barcodes for the preparation of 40 libraries: 4. When completed, press the "reate Worklist" button and save the file to the desktop. 20 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

21 Ligate adapters to the amplicons and purify Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "Ligation." 4. nter the number of reactions to be prepared. 5. If you are using ast Mode, enter "1" when prompted "nable ast Transfer?." Otherwise leave value as "0." 6. lick RUN. The run time is ~45 minutes (Standard) or 20 minutes (ast) for 96 reactions. 7. When the script is complete, seal the plate with Micromp dhesive ilm. Vortex the plate three times for 3 seconds each at setting 7 10, then centrifuge the plate briefly. 8. Place a Micromp ompression Pad on the plate, load the plate in the thermal cycler, and run the following program. Temperature Time min [1] min 10 old (for up to 1 hour) [1] or libraries from P samples, 60 min may increase yield. STOPPIN POINT Samples may be stored at 20. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 21

22 Option 1: qualize the library Option 1: qualize the library The Ion Library qualizer Kit (at. no ) provides a method for normalizing library concentration at ~100 pm without the need for quantitation. irst amplify the unamplified Ion mpliseq library, then capture the library on qualizer eads. fter elution of the equalized library, proceed directly to combining libraries and/or template preparation. Purify, elute, and amplify the library Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: Table 7 Reagent setup summary: Purify, elute, and amplify the library No. Position escription Labware ill volume [1] rid 1, Site 2 70% thanol 100-mL reservoir 35 ml rid 1, Site 3 Waste 100-mL reservoir mpty rid 2, Site 1, Wells 1 4 Platinum PR SuperMix ii (black cap) 2.0-mL tube 1.3 ml each [2] rid 2, Site 1, Well 5 mplification Primers [3] 0.5-mL tube 240 µl [2] rid 2, Site 2 MPure XP eads 25-mL reservoir 3.5 ml rid 10, Site 2 mplification plate (output) nduraplate reaction plate rid 10, Site 3 Ligated N plate (input) nduraplate reaction plate mpty rom previous reaction rid 16, Site 3 Plate magnet ynamag Side-96 n/a [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] Use a full tube(s) from a 96-LV kit to accommodate dead volumes. [3] See the Ion mpliseq N and RN Library Preparation User uide (Pub. no. MN ) for proper primer selection. 22 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

23 Option 1: qualize the library 2. ill a 100-mL trough with freshly prepared 70% ethanol and place it at rid 1, Site 2 (). 3. Place an empty 100-mL trough at rid 1, Site 3 (). 4. Place the tubes containing Platinum PR SuperMix igh idelity (black cap) in positions 1 4 of the chilled metal cooling block at rid 2, Site 1 (). Note: ll four tubes are required. 5. Place the tube containing the appropriate mplification Primers in position 5 of the chilled metal cooling block at rid 2, Site 1 (). Primer tube label Protocol compatibility qualizer Primers qualizer or gilent 2100 ioanalyzer / Qubit 2.0 luorometer 25X Library mplification Primers Library mplification Primer Mix qualizer or gilent 2100 ioanalyzer / Qubit 2.0 luorometer gilent 2100 ioanalyzer / Qubit 2.0 luorometer 6. ill a 25-mL trough with MPure XP eads and place it inside a 100-mL trough at rid 2, Site 2 (). 7. Place a new Micromp nduraplate reaction plate on the incubator at rid 10, Site 2 (). 8. Place a Micromp nduraplate reaction plate containing ligated N on the incubator at rid 10, Site 3 (). 9. Place the ynamag Side Magnet at rid 16, Site 3 (). 10. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "Library_mp." 4. nter the number of reactions to be prepared. 5. lick RUN. The run time is ~85 minutes for 96 reactions. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 23

24 Option 1: qualize the library 6. When the script is complete, seal the plate with a Micromp dhesive ilm, place a Micromp ompression Pad on the plate, load the plate in the thermal cycler, and run the following program: Note: Wash the qualizer eads (described in Wash the qualizer beads on page 24) while cycling, if necessary. Stage Temperature Time old 98 2 min 7 cycles sec 64 1 min old 10 old (for up to 1 hour) efore starting Wash the qualizer beads Warm all the reagents in the olon and Lung Kit ox 2 (4 ) to room temperature. Vortex and spin down all reagents before use. If not previously performed: 1. ring the qualizer eads to room temperature and mix thoroughly. Note: eads for multiple reactions may be prepared in bulk, and can be stored in qualizer Wash uffer at 4 for up to 6 months until use. fter 6 months, beads should be re-washed with an equal volume of qualizer Wash uffer. 2. or each reaction, pipet 3 μl of beads/reaction into a clean 1.5 ml tube and add 6 μl/reaction of qualizer Wash uffer. 3. Place the tube in a magnetic rack for 3 minutes or until the solution is completely clear. 4. arefully remove and discard the supernatant without disturbing the pellet. 5. Remove the tube/plate from the magnet, add 6 μl/reaction of qualizer Wash uffer, and pipet up and down to resuspend. 24 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

25 Option 1: qualize the library dd qualizer beads and wash Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: I Table 8 Reagent setup summary: dd qualizer eads and wash No. Position escription Labware ill volume [1] rid 1, Site 2 qualizer Wash uffer 100-mL reservoir 35 ml [2] rid 1, Site 3 Waste 100-mL reservoir mpty rid 2, Site 1, Well 1 (Standard Transfer Only) rid 2, Site 1, Well 2 (Standard Transfer Only) qualizer apture 2.0-mL tube 1.2 ml [2] Washed qualizer eads 1.5-mL tube 750 µl rid 2, Site 3 qualizer lution uffer 25-mL reservoir 12 ml [2] rid 10, Site 1 (ast Transfer Only) qualizer apture and eads rid 10, Site 2 qualizer Library (Output) rid 10, Site 3 mplified Library Plate (input) nduraplate reaction plate nduraplate reaction plate nduraplate reaction plate apture: 150 µl/well in column 6 Washed eads: 92 µl/well in column 8 mpty rom previous reaction I rid 16, Site 3 Plate Magnet ynamag Side-96 n/a [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] Use a full tube(s) from an Ion Library qualizer Kit to accommodate dead volumes. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 25

26 Option 1: qualize the library 2. dd the following reagents according to the transfer mode selected: Transfer Mode Reagent Setup Standard 1. Place the tube containing qualizer apture in position 1 of the chilled metal cooling block at rid 2, Site 1 (). 2. Place the tube containing washed qualizer eads in position 2 of the chilled metal cooling block at rid 2, Site 1 (). ast 1. liquot qualizer apture into all wells of column 6 and washed qualizer eads into all wells of column 8 in a Micromp nduraplate reaction plate and place it on the incubator at rid 10, Site 1 (). This plate can be stored at 4 for later use. 3. ill a 100-mL trough with qualizer Wash uffer and place it at rid 1, Site 2 (). 4. Place an empty 100-mL trough at rid 1, Site 3 (). 5. ill 25-mL trough with qualizer lution uffer and place it inside a 100-mL trough at rid 2, Site 3 (). 6. Place a new Micromp nduraplate reaction plate on the incubator at rid 10, Site 2 (). 7. Place a Micromp nduraplate reaction plate containing amplified library (from Purify, elute, and amplify the library on page 22) on the incubator at rid 10, Site 3 (). 8. Place the ynamag Side Magnet at rid 16, Site 3 (I). 9. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "qualizer." 4. nter the number of reactions to be prepared. 5. lick RUN. The run time is ~100 minutes (Standard) or 85 minutes (ast) for 96 reactions. 6. When the script is complete, the plate at rid 10, site 3 contains the qualized library. Proceed immediately to template preparation, or combine and/or store the library as described in the Ion mpliseq N and RN Library Preparation User uide (Pub. no. MN ). Note: The final concentration of each qualized library is ~100 pm. 26 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

27 Option 1: qualize the library Store libraries Libraries may be stored at 4 8 for up to 1 month. or longer term storage, store at -20. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 27

28 Option 2: Purify and quantify the unamplified library by qpr Option 2: Purify and quantify the unamplified library by qpr Purify and elute the unamplified Ion mpliseq library, then determine the concentration by qpr with the Ion Library Quantitation Kit (at. no ). fter quantitation, determine the dilution factor that results in a concentration of ~100 pm, which is suitable for template preparation using an Ion template kit. Note: The Ion Library Quantitation Kit may also be used to quantify libraries that have been amplified using the procedure described in "lute and amplify the library" on page 24. Purify the unamplified library Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: Table 9 Reagent setup summary: Purify the unamplified library No. Position escription Labware ill volume [1] rid 1, Site 2 70% thanol 100-mL reservoir 35 ml rid 1, Site 3 Waste 100-mL reservoir mpty rid 2, Site 2 MPure XP eads 25-mL reservoir 3.5 ml rid 2, Site 3 Low T 25-mL reservoir 6 ml rid 10, Site 2 inished library (output) rid 10, Site 3 Ligated N plate (input) nduraplate reaction plate nduraplate reaction plate mpty rom previous reaction rid 16, Site 3 Plate Magnet ynamag Side-96 n/a [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. 28 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

29 Option 3: Quantify the amplified library with Qubit 2.0 luorometer or gilent 2100 ioanalyzer 2. ill a 100-mL trough with freshly prepared 70% ethanol and place it at rid 1, Site 2 (). 3. Place an empty 100-mL trough at rid 1, Site 3 (). 4. ill a 25-mL trough with MPure XP eads and place it at rid 2, Site 2 (). 5. ill a 25-mL trough with Low T and place it at rid 2, Site 3 (). 6. Place a new Micromp nduraplate reaction plate on the incubator at rid 10, Site 2 (). 7. Place a Micromp nduraplate reaction plate containing ligated N (from Ligate adapters on page 19) on the incubator at rid 10, Site 3 (). 8. Place the ynamag Side Magnet at rid 16, Site 3 (). 9. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "qpr_purification." 4. nter the number of reactions to be prepared. 5. lick RUN. The run time is ~85 minutes for 96 reactions. 6. When the script is complete, the plate at rid 10, Site 2 contains the purified library. Prepare a 100-fold dilution of the library by removing 2 μl of supernatant and combining with 198 μl of Nuclease-ree Water for quantitation. 7. Proceed to "Quantify library by qpr and calculate dilution factor," in the Ion mpliseq N and RN Library Preparation uide (Pub. no. MN ). Option 3: Quantify the amplified library with Qubit 2.0 luorometer or gilent 2100 ioanalyzer Ion mpliseq libraries must be amplified prior to quantitation with the Qubit 2.0 luorometer or gilent 2100 ioanalyzer. Note: Library amplification is required for this method, to enrich amplifiable material and obtain sufficient material for accurate quantification using these instruments. The Ion Library Quantitation Kit may also be used to quantify amplified libraries. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 29

30 Option 3: Quantify the amplified library with Qubit 2.0 luorometer or gilent 2100 ioanalyzer mplify the library Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by-step instructions provided below: Table 10 Reagent setup summary: mplify the library No. Position escription Labware ill volume [1] rid 1, Site 2 70% thanol 100-mL reservoir 35 ml rid 1, Site 3 Waste 100-mL reservoir mpty rid 2, Site 1, Wells 1 4 rid 2, Site 1, Well 5 Platinum PR SuperMix ii (black cap) 2.0-mL tube 8 ml mplification Primers [2] 0.5-mL tube 6 ml rid 2, Site 2 MPure XP eads 25-mL reservoir mpty rid 10, Site 2 mplification plate (output) nduraplate reaction plate rom previous reaction rid 10, Site 3 Ligated N plate (input) nduraplate reaction plate on Micromp Splash-free 96-well ase rid 16, Site 3 Plate magnet ynamag Side-96 n/a [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. [2] See the Ion mpliseq N and RN Library Preparation User uide (Pub. no. MN ) for proper primer selection. mpty 2. ill a 100-mL trough with freshly prepared 70% ethanol and place it at rid 1, Site 2 (). 3. Place an empty 100-mL trough at rid 1, Site 3 (). 4. Place the tubes containing Platinum PR SuperMix ii in positions 1 4 of the chilled metal cooling block at rid 2, Site 1 (). Note: ll four tubes are required. 30 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

31 Option 3: Quantify the amplified library with Qubit 2.0 luorometer or gilent 2100 ioanalyzer 5. Place the tube containing the appropriate library mplification Primers in position 5 of the chilled metal cooling block at rid 2, Site 1 (). Primer tube label Protocol compatibility qualizer Primers qualizer or gilent 2100 ioanalyzer / Qubit 2.0 luorometer 25X Library mplification Primers Library mplification Primer Mix qualizer or gilent 2100 ioanalyzer / Qubit 2.0 luorometer gilent 2100 ioanalyzer / Qubit 2.0 luorometer 6. ill a 25-mL trough with MPure XP eads and place it inside a 100-mL trough at rid 2, Site 2 (). 7. Place a new Micromp nduraplate reaction plate on the incubator at rid 10, Site 2 (). 8. Place a Micromp nduraplate reaction plate containing ligated N on the incubator at rid 10, Site 3 (). 9. Place the ynamag Side Magnet at rid 16, Site 3 (). 10. Replace the tips boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "Library_mp." 4. nter the number of reactions to be prepared. 5. lick RUN. The run time is ~85 minutes for 96 reactions. 6. When the script is complete, seal the plate with a Micromp dhesive ilm. 7. Place a Micromp ompression Pad on the plate, load the plate in the thermal cycler and run the following program: Stage Temperature Time old 98 2 min 5 cycles sec 64 1 min old 10 old STOPPIN POINT Samples may be stored at 20. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 31

32 Option 3: Quantify the amplified library with Qubit 2.0 luorometer or gilent 2100 ioanalyzer Purify the amplified library Setup the worktable 1. Setup the worktable as shown. or more details, see the step-by step instructions provided below: Table 11 Reagent setup summary: Purify the amplified library No. Position escription Labware ill volume [1] rid 1, Site 2 70% thanol 100-mL reservoir 35 ml rid 1, Site 3 Waste 100-mL reservoir mpty rid 2, Site 2 MPure XP eads 25-mL reservoir 8 ml rid 2, Site 3 Low T 25-mL reservoir 6 ml rid 10, Site 2 inished library (output) nduraplate reaction plate mpty rid 10, Site 3 mplified library (input) nduraplate reaction plate rom previous reaction rid16, Site 2 lean-up plate nduraplate reaction plate on Micromp Splash-ree 96-Well ase mpty rid 16, Site 3 Plate magnet ynamag Side-96 n/a [1] or 96 reactions. If running fewer than 96 samples, adjust volumes accordingly. 2. ill a 100-mL trough with freshly prepared 70% ethanol and place it at rid 1, Site 2 (). 3. Place an empty 100-mL trough at rid 1, Site 3 (). 4. ill a 25-mL trough with MPure XP eads and place it inside a 100-mL trough at rid 2, Site 2 (). 32 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

33 Quantify the library using the Qubit 2.0 luorometer 5. ill at 25-mL trough with Low T and place it inside a 100-mL trough at rid 2, Site 3 (). 6. Place a new Micromp nduraplate reaction plate on the incubator at rid 10, Site 2 (). 7. Place a Micromp nduraplate reaction plate containing amplified N (from mplify the library on page 30) on the incubator at rid 10, Site 3 (). 8. Place a new Micromp nduraplate on a Micromp Splash-ree 96-Well ase at rid 16, Site 2 (). 9. Place the ynamag Side Magnet at rid 16, Site 3 (). 10. Replace the tip boxes as needed. Run the script 1. Open VOware. 2. lick Run an existing script. 3. Select the script "Library_mp_Purification." 4. nter the number of reactions to be prepared. 5. lick RUN. The run time is ~85 minutes for 96 reactions. 6. Once the script is complete, the plate at rid 10, Site 2 contains the purified library. Quantify the library using the Qubit 2.0 luorometer nalyze 10 μl of each amplified library using the Qubit 2.0 luorometer (at. no. Q32866) and the Qubit dsn S ssay Kit. mplified libraries typically have concentrations of ng/ml. or more information, see the Qubit dsn S ssay Kits User uide. (Pub. no. MN ) 1. etermine the amplified library concentration: a. Make a 1:200 working dilution of Qubit dsn S reagent using the Qubit dsn S uffer. b. ombine 10 μl of the amplified Ion mpliseq 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 luorometer. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 33

34 Quantify the library using gilent 2100 ioanalyzer Instrument e. alculate the concentration of the undiluted library by multiplying by 20. This can be calculated automatically using the alculate Stock oncentration button and inputting 10 μl as the sample volume. 2. ased on the calculated library concentration, determine the dilution that results in a concentration of ~100 pm (15 ng/ml for amplicons up to 225 bp, 22 ng/ml for amplicons up to 275 bp) or example, with a P-compatible bp design: The library concentration is 450 ng/ml. The dilution factor is 450 ng/ml divided by 15 ng/ml = 30. Therefore, 10 μl of library mixed with 290 μl of Low T (1:30 dilution) yields approximately 15 ng/ml (~100 pm). 3. ilute library to ~100 pm as described and proceed to combining libraries or template preparation. Quantify the library using gilent 2100 ioanalyzer Instrument nalyze 1 μl of amplified library on the gilent 2100 ioanalyzer instrument with the gilent igh Sensitivity N Kit (at. no ). mplicon libraries should have multiple peaks in the bp size range. mplified libraries typically have concentrations of 1,000 5,000 pm. If the library concentration is over 20,000 pm, dilute the library 1:10 and repeat the quantification to obtain a more accurate measurement. 1. etermine the molar concentration of the amplified library using the ioanalyzer software. nsure that the upper and lower marker peaks are identified and assigned correctly. ollow the manufacturer s instructions to perform a region analysis (smear analysis). riefly: a. Select the ata icon in the ontexts 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. orrect the baseline if needed. c. The molarity is automatically calculated and displayed in the table in pmol/l (pm). 2. ased on the calculated library concentration, determine the dilution that results in a concentration of ~100 pm. or 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 T (1:30 dilution) yields approximately 100 pm. 3. ilute library to ~100 pm as described and proceed to combining libraries or template preparation. 34 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

35 (Optional) ombine amplicon libraries (Optional) ombine amplicon libraries There are multiple strategies for combining Ion mpliseq libraries, as described in Ion mpliseq N and RN Library Preparation User uide (Pub. no. MN ). Store libraries Libraries may be stored at 4 8 for up to 1 month. or longer term storage, store at -20. Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation 35

36 ocumentation and support ocumentation and support ustomer and technical support Ion contact information Visit for the latest in services and support, including: Worldwide contact telephone numbers Product support, including: Product Qs Software, patches, and updates Order and web support Product documentation, including: User guides, manuals, and protocols ertificates of nalysis Safety ata Sheets (SSs; also known as MSSs) Note: or SSs for reagents and chemicals from other manufacturers, contact the manufacturer. Web site: lifetechnologies.com/iontorrent Ion community: ioncommunity.lifetechnologies.com Support Phone numbers In North merica: 1-87-SQUN ( ) Outside of North merica: Limited product warranty Life Technologies orporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' eneral Terms and onditions of Sale found on Life Technologies' website at If you have any questions, please contact Life Technologies at support. 36 Prepare Ion mpliseq Libraries using the Tecan reedom VO NS Workstation

37

38 The information in this guide is subject to change without notice. ISLIMR TO T XTNT LLOW Y LW, LI TNOLOIS N/OR ITS ILIT(S) WILL NOT LIL OR SPIL, ININTL, INIRT, PUNITIV, MULTIPL, OR ONSQUNTIL MS IN ONNTION WIT OR RISIN ROM TIS OUMNT, INLUIN YOUR US O IT. Important Licensing Information These products may be covered by one or more Limited Use Label Licenses. y use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. Trademarks ll trademarks are the property of Thermo isher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. gilent and ioanalyzer are trademarks of gilent Technologies, Inc. gencourt and MPure are trademarks of eckman oulter, Inc. Nanorop is a trademark of Nanorop Technologies, LL. reedom VO is a registered trademark of Tecan roup, Ltd Thermo isher Scientific Inc. ll rights reserved. or support visit lifetechnologies.com/support or techsupport@lifetech.com lifetechnologies.com 30 March 2015