user’s guide illumina truseq dna - perkinelmer · user’s guide illumina truseq dna library...

USER’S GUIDE ILLUMINA

TRUSEQ DNA LIBRARY PREPARATIONON THE SCICLONENGS LIQUID HANDLER

Compatible with: Illumina TruSeq™ DNA Sample Prep v2 kits Sciclone® NGS Workstation with Maestro™ 6.0 Sciclone NGSx Workstation with Maestro 6.2

2

Introduction 3

Maestro TruSeq™ DNA Library Prep Workflow Overview 4-5

Required Materials and Reagents 6

Reagents 6

General Laboratory Equipment and Supplies 6

Sciclone NGS Workstation Accessories 6

Consumables 7

Running the Maestro TruSeq™ DNA Application 7

Sample Preparation 7

“TruSeqDNA Library Prep” Run Preparation Steps 7-10

PCR Enrichment 10

“TruSeq Post-PCR SPRI” Run Preparation Steps 10-11

Library QC and Storage 11

Expected Results 11

Appendix A: Step-by-Step Guide to the “TruSeqDNA Library Prep” Application 12-13

Appendix B: Step-by-Step Guide to the “TruSeq Post-PCR SPRI” Application 13-14

Table of Contents

Illumina TruSeq™ DNA User Guide for the Sciclone NGS Workstation

www.perkinelmer.com/ScicloneNGS 3


Introduction

Preparation of DNA samples for cluster generation and sequencing on the Illumina, Inc. platform requires a series of manipulations to efficiently ligate appropriate indexed adapters onto DNA fragments to produce paired-end libraries. Automating the process has the advantage of avoiding sample tracking errors and reducing sample-to-sample variability while dramatically increasing throughput. The Maestro-based TruSeq™ DNA Application from PerkinElmer provides a pre-programmed solution for Illumina TruSeq™ DNA Library Preparation on the Sciclone NGS Workstation from PerkinElmer.

Figure 1. Overview of the steps for TruSeq™ DNA library preparation on the Sciclone NGS Workstation.

Maestro TruSeq™ DNA Workflow

4


Maestro TruSeq™ DNA Workflow Overview

Set-up Run Time (Including Maestro Application Time Thermocycler Steps)

1 “TruSeqDNA Library Prep” 1 hour 4.5 hours

2 “TruSeq Post-PCR SPRI” 10 min 45 min

The Maestro Application for TruSeq™ DNA sample preparation is a validated process for library preparation from fragmented DNA samples that follows the steps outlined in Figure 1. Samples are processed in 96-well PCR plates, and the number of samples to process (1 to 12 columns of 8 samples each) is selected at the start of each run. Pre-set tip-tracking utilities written into the application, guide the instrument to pick up appropriate numbers of tips and refill/replace tip boxes as needed. INHECO temperature blocks installed on the Sciclone NGS Workstation deck allow for appropriate 4 ˚C storage of reagents and heated incubations of reactions. Reaction mixes are pre-arrayed prior to addition to sample to ensure equal incubation times across the sample plate. Adapter indexing patterns may be executed during the run by arraying 1-24 different adapters to appropriate well locations as specified by the user in an Microsoft® Excel® workbook. Easy-to-follow user interfaces guide the reagent and deck setup process and prompt the user for any necessary interventions.

Two independent Maestro Applications are used in the 1-day TruSeq DNA Workflow: “TruSeqDNA Library Prep” and “TruSeq Post-PCR SPRI”. The Maestro TruSeq™ DNA sample preparation process differs from the Illumina protocol in several important aspects. First, a single aliquot of AMPure® XP beads is used per sample throughout the post-end repair, A-tailing, and ligation steps. The DNA in the sample is driven on and off the beads via changes in PEG and NaCl concentrations. This strategy increases yield by limiting the number of times samples are transferred to new wells/plates. Second, the volumes/concentrations of some reagent additions have been reduced from the Illumina-recommended volumes/concentrations. These reductions allow for the necessary overage volume during automated pipetting steps without significantly affecting yields. Reducing the concentration of adapters in the ligation step helps prevent excess adapter-dimer ligation. Third, purification and size selection of ligation products is complete with a single AMPure® XP bead cleanup, rather than gel-based size selection. The AMPure® XP cleanup is amenable to automation and high throughput sample processing, and has proved effective at reducing the adapter-dimer concentration prior to PCR enrichment of the library fragments. The modifications summarized above have led to the development of a robust automated procedure (end repair through post-PCR cleanup) for up to 96 samples that can be completed in 6 hours.

5www.perkinelmer.com/ScicloneNGS


Figure 2. “TruSeqDNA Library Prep” and “TruSeq Post-PCR SPRI” Application steps.

6


Sciclone NGS Workstation Accessories

Accessory PerkinElmer Part No.

Agencourt 96-ring Magnet CLS128316

INHECO 384-well plate adapter CLS128373

INHECO 96-well adapters (3) CLS128372

INHECO 96-well adapter/shaker CLS100852

Required Materials and Reagents

Reagents

Reagent Vendor and Part No.

TruSeq™ DNA Sample Illumina, Inc. Preparation Kit (48 samples) With Set A adapters – 15012999 With Set B adapters – 15013001

Nuclease-Free Water Various

AMPure® XP Beads Beckman Coulter – A63881 (60 mL)

100% EtOH Sigma-Aldrich E7023

50% PEG 8000 Sigma-Aldrich 83271

5 M NaCl Sigma-Aldrich S5150

Note: TruSeq™ DNA kits supply enough reagents to process 48 samples. If kits are used in multiple runs with <48 samples per run, volumes of specific reagents may become limiting. Care should be taken to avoid freeze-thaw cycles with all TruSeq™ reagents. Reagents should be aliquoted appropriately if planning to use the kit for more than 2 independent runs.

Note: The Maestro “TruSeqDNA Library Prep” Application is not currently written to process samples with the Illumina-supplied End Repair Control, A-Tailing Control, or Ligase Control reagents. These kit components will not be used.

General Laboratory Equipment and Supplies

Equipment Supplier

Microfuge Various

Vortexer Various

200 µL Multichannel pipettor and appropriate barrier tips Various

1000 µL Multichannel pipettor and appropriate barrier tips Various

Covaris S2 or E210 System and appropriate tubes Covaris

Microplate centrifuge Various

Thermocycler** Bio-Rad Laboratories (MJ Research) DNA Engine PTC-200, or equivalent

Plate Seals Various – compatible with Thermocycler and freezer storage

PerkinElmer LabChip® GX or Agilent Technologies Bioanalyzer with appropriate chips and reagents Various

** The standard sample plates used in this application are fully skirted Bio-Rad Hard-Shell® 96-PCR plates. Please check if your thermocycler is compatible with this plate type. If it is not, please contact your PerkinElmer Field Application Scientist to discuss modifications to the application to support semi-skirted PCR plates.



Consumables

PerkinElmer No. Used Consumable Description Part No. Vendor and Part No. per Run

PCR Plates 96-well PCR Plate, Bio-Rad Hard-Shell®, Full Skirt CLS127737 Bio-Rad HSP-9631 13

Boxed Tips Pipette Tip 150 µL, Art, Box, 10-96 Sterile Racks CLS111426 PerkinElmer 20 (96 samples)

Deepwell Plates Deepwell-96 POS, Square 2.0 mL Well, Polypro, CLS133355 Seahorse Bioscience 201379-100 2 Seahorse Bioscience

Reservoir- 12-Column Pyramid Bottom, 290 mL CLS128477 Seahorse Bioscience 201250-100 2 Deepwell Seahorse Bioscience

Lids 946 Lid – Universal, Robotic Friendly, Polystyrene CLS112785 Seahorse Bioscience 200856-100 8

384-well Plates Microplate 384-well, Round Bottom, CLS134399 Corning, Inc. 3672 1 Polypropylene (Pkg. 10)

Running the Maestro TruSeq™ DNA Applications

Please read and familiarize yourself with all steps described in this section prior to beginning the run. For best results, the entire process should be completed in one day. Allow 6-7 hours to complete all steps, including reagent distribution, deck setup, end repair, A-tailing, ligation, PCR enrichment, and post-PCR cleanup of samples.

Sample Preparation

Genomic DNA should be fragmented to an average size of 300-400 bp on a Covaris S2 or E210 instrument as described in the Illumina TruSeq™ DNA Sample Preparation Guide (#15005180). Samples sheared to a smaller size will require alternative processing (see Step 4). Samples should be presented for the Maestro “TruSeqDNA Library Prep” run as up to 1 µg sheared DNA in 50 µL TE, low TE, RSB, or water in a Bio-Rad Hard-Shell® 96-well PCR plate.

“TruSeqDNA Library Prep” Run Preparation Steps

Note: AMPure® XP beads should be warmed at Room Temperature for about 30 min before use. They may be taken out of 4 ˚C storage before beginning. Do not thaw TruSeq™ reagents until steps 1 and 2 have been completed.

If necessary, boot up the system by first starting the Sciclone NGS Workstation and the INHECO units, then starting the PC controller.

1. Modify the Maestro “TruSeqDNA Library Prep Workbook” to Specify the Number of Samples to Run and the Adapter Index Pattern

The workbook must be located in the filepath: C:\Program Data\CaliperLS\Maestro\Workbooks\ and must have the name “TruSeq DNA Library Prep Workbook.xls”. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run.

8


The sample number must be set by indicating the number of columns to run in the worksheet titled “TruSeqDNA Reagent Plates”. The Maestro application only processes full columns of 8 samples each. Running more than 6 columns of samples will require 2 TruSeq™ DNA reagent kits, as each kit contains reagents for up to 48 samples.

The adapter index pattern is set by designating the appropriate adapter numbers to each well ID in column B of the spreadsheet titled “Indexing”. Up to 24 different adapters may be used. Illumina supplies 20 µL per tube of each adapter, allowing up to 8 samples per adapter when diluting adapter 1:2 with Resuspension Buffer (RSB).

Enter a number from 1-27 in the “Index Well” column for each of the samples to be run. The TruSeq™ adapters are numbered from ID001 to ID027, with adapters ID017, ID024 and ID026 omitted. If desired, custom adapters can be used and the indexed adapter names in cells I4-I30 may be changed. The chart on the right will automatically update the number of samples using each adapter index. This information will be passed into the worksheet for “TruSeq DNA Reagent Plates” to modify the volume necessary for each adapter index mix.

After modifying entries for the number of columns to process and the adapter index pattern, the spreadsheet will update the recipes for the reagent mixes and the appropriate volumes for the reagent plates. Save the modified spreadsheet with its original name in its original file path. Print the “TruSeqDNA Reagent Plates” spreadsheet to use as a guide while setting up reagents.

2. Start the Maestro “TruSeqDNA Library Prep” Application

Launch the Maestro software and open the “TruSeqDNA Library Prep” Application. Start the run by selecting the play button. If running in Editor Mode, be sure to start the Main Method.

Note: When the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the library preparation steps. Starting the application prior to thawing and diluting reagents ensures that the cold blocks are pre-chilled and ready for on-deck reagent storage.

Ensure that the INHECO units have the correct adapters for the run: Position A3 384-well Plate Adapter Position A4 96-well PCR Plate Adapter Position D2 96-well PCR Plate Adapter Position D4 96-well PCR Plate Shaker Adapter

Verify that the INHECO units for positions A3 and A4 are set to 4 ˚C and are cooling.

Figure 3. “TruSeqDNA Reagent Plates” spreadsheet in the “TruSeqDNA Library Prep” workbook.

Figure 4. “Indexing” spreadsheet in the “TruSeqDNA Library Prep” Workbook.

Figure 5. “TruSeqDNA Reagent Plates” spreadsheet in the “TruSeqDNA Library Prep” Workbook.



3. Thaw the TruSeq™ Reagents and Place on Ice

Care should be taken to avoid freeze-thaw cycles with all TruSeq™ reagents. Reagents should be aliquoted appropriately if planning to use a kit for more than 2 independent runs. It is helpful to place the tubes containing RSB and End Repair Mix in water to speed the thawing process.

4. Prepare the 80% EtOH Reservoir, PEG Plate, RSB Plate, and AMPure® XP Bead Plate

Make 100 mL fresh 80% EtOH solution by diluting 80 mL 100% Ethanol with 20 mL nuclease-free molecular biology grade water. Pour 100 mL fresh 80% EtOH into a Seahorse Bioscience Deepwell reservoir, cover with a lid and store at room temperature.

Use the “TruSeqDNA Reagent Plates” spreadsheet as a guide for setting up the plates.

If necessary, make a 20% PEG/2.5M NaCl solution and 20% PEG/3M NaCl solution. These solutions may be made in advance and stored at room temperature.

Using a multichannel pipettor, aliquot 30 µL 20% PEG/2.5M NaCl solution per well into a Bio-Rad Hard-Shell® PCR plate for each column of samples to be run. Label the plate, cover with a lid, and store at room temperature.

Using a multichannel pipettor, aliquot 120 µL Resuspension Buffer (RSB) per well into a Bio-Rad Hard-Shell® PCR plate for each column of samples to be run.

Note: If it is desired to retain smaller DNA fragments (150 to 250 bp) in the post-ER SPRI® cleanup, then AMPure™ XP beads will need to undergo a bead buffer exchange step to substitute the existing buffer on the AMPure™ XP beads with an equal volume of 20% PEG/3M NaCl solution. The higher salt content in the bead buffer will allow recovery of shorter fragments (<250 bp) in the post End Repair SPRI® cleanup step.

To exchange the bead buffer, thoroughly resuspend AMPure™ XP beads (warmed to room temperature) by inverting/rotating the bottle. Transfer the total volume of beads needed (plus 10% overage volume) to one or more 2 mL eppendorf tubes or to a 15 mL falcon tube. Pellet the beads using a magnet. Remove the buffer from the beads and replace it with an equal volume of 20% PEG/3M NaCl. Thoroughly resuspend the beads by inverting/rotating the tube(s).

Using a multichannel pipettor, aliquot 105 µL AMPure™ XP beads per well into a Bio-Rad Hard-Shell® PCR plate for each column of samples to be run. Cover the plate with a lid and store at room temperature.

Inspect all plates to ensure that air has not been trapped in the wells. If necessary, spin the plates briefly to bring reagents to the bottom of the wells.

5. Make the reaction master mixes and adapter mixes

The following mixes should be prepared according to the recipes in the “TruSeq DNA Reagent Plates” spreadsheet: End-Repair Mix, A-Tailing Mix, Ligation Mix, Adapter Mixes (up to 24), PCR Mix.

Gently mix and centrifuge each Illumina supplied reagent tube prior to use. After mixing the appropriate volumes of reagents in nuclease-free tubes, mix by inverting and centrifuge again to collect all liquid at the bottoms of the tubes.

Care should be taken to pipette accurately, as some reagents are viscous and minimal overage volumes are used. Keep the reaction mixes on ice.

6. Aliquot the reaction mixes into plates

The Bio-Rad Hard-Shell® PCR plate should be filled with the specified volumes of A-tailing Mix, Ligase Mix, Stop Ligase Buffer, Adapter Mixes, End Repair Mix and PCR Master Mix in the specified wells. Aliquot the reaction mixes to the reagent plate in the wells indicated on the “TruSeq DNA Reagent Plates” spreadsheet. Keep the plates on ice while pipetting to keep the reagents cold. Pipette carefully into the bottom of the wells and avoid trapping air or creating bubbles. If necessary, spin the plates briefly in a plate centrifuge to ensure all reagents are at the bottom of the wells. Label the plate, cover with a lid and store on ice or at 4 ˚C.

7. Set Up the Deck of the Sciclone NGS Workstation

Confirm that the Maestro software has correctly read the workbook and is set to run the correct number of columns. If the incorrect number of columns is indicated, modify and save the Microsoft® Excel® workbook as described in Step 1, then start the application again from the Main Method.

Step through the pictures, placing the indicated consumables/prepared plates in the indicated locations. Be sure to check whether a lid is needed for each plate or reservoir. Place new tip boxes in the indicated locations.

8. Run Library Preparation Steps

Confirm that the deck setup matches the final picture in the setup window. Selecting “Finished” will prompt the application to begin the library prep protocol.

The Maestro “TruSeqDNA Library Prep” Application will automatically proceed through End Repair, A-tailing, Ligation, and PCR setup steps as indicated in the flowchart in Figure 2 (Page 5). While the application is running, the green light at the top of the instrument will blink. If there is a problem with the run, the light will change to yellow and an alarm will sound to indicate that user intervention is necessary. See Appendix A (Page 12) for a Step-by-Step guide of the Maestro “TruSeqDNA Library Prep” Application on the Sciclone NGS Workstation.

10


When the PCR setup is complete, the application will pause and show a message indicating that the PCR plate should be sealed and placed on a thermocycler for the amplification step. Close the dialog box to complete the run and shut down the leg lights and INHECO temperature controls.

Note: The RSB and 80% EtOH plates may be retained for use in the “TruSeq Post-PCR SPRI” cleanup.

PCR Enrichment

Illumina recommends the following program for amplification of libraries prepared from 1 µg input DNA. The number of cycles may need to be modified according to the amount of input DNA in the sample. Use a heated lid to prevent condensation.

PCR: 98 ˚C for 30 seconds 10 cycles of: 98 ˚C for 10 seconds 60 ˚C for 30 seconds 72 ˚C for 30 seconds 72 ˚C for 5 minutes Hold at 10 ˚C

“TruSeq Post-PCR SPRI” Run Preparation Steps

The “TruSeq Post-PCR SPRI” cleanup step is provided as a separate Maestro application. If desired, the user may designate a different liquid handler for post-PCR sample processing to avoid any possible cross-contamination of pre-amplification samples. “TruSeq Post-PCR SPRI” cleanup applications are available for both the Sciclone NGS Workstation and the Zephyr NGS Workstations. The instructions here are for running the “TruSeq Post-PCR SPRI” application on the Sciclone NGS Workstation.

Note: AMPure® XP beads should be warmed at Room Temperature for about 30 min before use. They should be taken out of 4 ˚C storage when the PCR Enrichment step is started. If the RSB remaining in the Bio-Rad Hard-Shell® 96-well plate has not been saved at the completion of the library prep, it will also be necessary to thaw the RSB buffer from the Illumina library prep kit.

1. Modify the “TruSeq Post-PCR SPRI” Workbook

Enter the number of columns to be processed and save the workbook. The workbook must be located in the filepath: C:\ProgramData\Caliperls\Maestro\Workbooks\ and must have the name “TruSeq Post-PCR SPRI Workbook.xls”. If the file is moved or the name is changed, Maestro will not be able to find the information necessary to begin the run.

2. Start the Maestro “TruSeq Post-PCR SPRI” Application

Launch the Maestro software and open the “TruSeq Post-PCR SPRI” application. Start the run by selecting the play button. If running in Edit mode, be sure to start the Main Method.

Note: When the run is started, the instrument will complete all initialization steps for the hardware and the specific application. The run will automatically pause and prompt the user to set up the deck and confirm proper setup prior to beginning the library preparation steps.

3. Prepare the SPRI® Beads Plate and RSB Plate

Aliquot 55 µL of AMPure® XP beads per well in a Bio-Rad Hard-Shell® PCR plate. Ensure that the beads are at room temperature and well mixed prior to pipetting.

If the 96-well Bio-Rad Hard-Shell® PCR plate containing RSB used in the “TruSeqDNA Library Prep” application is saved at the completion of the library prep steps, the same plate may be used for RSB during the post-PCR cleanup. Confirm that enough RSB remains in the wells (30 µL plus >5 µL overage volume in each well), or set up a new plate as indicated in the workbook.

4. Prepare 80% Ethanol Reservoir

If the 80% EtOH reservoir used in the “TruSeqDNA Library Prep” run has been retained, it can be used for this run. Otherwise, make 50 mL fresh 80% EtOH solution by diluting 40 mL 100% Ethanol with 10 mL nuclease-free molecular biology grade water. Pour 50 mL fresh 80% EtOH into a Seahorse Bioscience deepwell reservoir and cover with a lid.

5. Setup the Sciclone Deck

Confirm that the software has correctly read the workbook and is set to run the correct number of columns. Step

Figure 6. “TruSeq Post-PCR SPRI” Workbook.



Figure 8. Representative LabChip GX data tracings of 4 individual libraries from the data shown in Figure 7.

Figure 9. Total yield of library calculated from the LabChip GX data shown in Figure 7. Samples A1 through D6: 1000 ng input DNA. Samples E1 through H6: 500 ng input DNA.

Figure 7. LabChip GX data from dilutions of 48 libraries prepared from 1000 ng or 500 ng DNA (Human Genomic DNA, Coriell #NA19240, sheared to 250 bp). Samples marked “1:1” were prepared with 1:1 dilution of adapter in RSB. Samples marked “undiluted” were prepared with undiluted adapters.

through the pictures, placing the indicated consumables/prepared plates in the indicated locations. Be sure to check whether a lid is needed for each plate or reservoir. Place new tip boxes in the indicated locations.

Note: When the “TruSeq Post-PCR SPRI” application is started, the variables used for tip tracking are reset. The run must be started with new, full tip boxes in the indicated positions, as Maestro will not retain tip tracking information from the library prep run.

6. Run the “TruSeq Post-PCR SPRI” Steps

Confirm that the deck setup matches the final picture in the setup window. Selecting “Finished” will prompt the application to begin the library prep steps. See Appendix B (Page 13) for a Step-by-Step guide to the Sciclone steps of the “TruSeq Post-PCR SPRI” application.

When the run is complete, the application will pause and show a message indicating that the sample plate should be sealed and stored appropriately. Close the dialog box to

complete the run and shut down the leg lights and INHECO temperature controls.

Library QC and Storage

Seal the library plate and store at -20 ˚C for up to 7 days, or proceed directly into library validation prior to storing.

The LabChip® GX Automated Electrophoresis System may be used to check the size distribution of fragments in the amplified library and estimate the concentration of fragments in the appropriate size range. Make a 1:25 dilution of library into molecular biology grade water in a Bio-Rad Hard-Shell® skirted 96-PCR plate. Mix well by pipetting up and down, and spin the plate to remove any bubbles. Run the samples on the LabChip GX System using a High Sensitivity DNA chip and kit according to the standard LabChip protocol.

Additional/alternative validation and quantification, including qPCR quantification, should be completed according to the user’s standard practices.

Expected Results

12


Appendix A

Step-by-Step Guide to “TruSeqDNA Library Prep” Application

Step 1: Broadcast End Repair

1. Move lid on Reagent Plate A4 to C4

2. Load tips (single column) and transfer 45 µL of ERP from Col 8-10 of the Reagent Plate (A4) to the PCR Plate on CPAC in D2, Eject tips.

3. Move lid C4 to Reagent Plate A4.

Step 2: End Repair

1. Move lid from Sample Plate (D4) to C4.

2. Load tips. Transfer 40 µL ERP (D2) to sample plate (D4), Mix, Eject tips.

3. Dispose of ERP plate (D2).

4. Move lid from C4 to Sample Plate D4.

5. Incubate End Repair Reaction for 30 mins at 30 ˚C.

During Incubation Pre-Broadcast Adapters to 384-well Plate

a. Move lid A4 to C4. Move lid A3 to A2.

b. Load tips (single column) and eject to empty tip box at C5. Load single tip from C5 for each adapter transfer.

c. Broadcast adapters one at a time. Aspirate from Reagent Plate A4 col 4-6 and multi-dispense to all appropriate wells in Q1 of 384-well at A3. Eject tip. Repeat for all adapters.

d. Move lid C4 to Reagent Plate (A4). Move lid A2 to 384-well Plate (A3).

6. Remove Lid from Sample Plate (D4) to A2. Move Sample Plate from D4 to C4.

7. Dispose of Lid on 2 PCR Plates at B2. Move top plate (SPRI® Bead Plate) from B2 to D4.

8. Remove any remaining tips from Tip Box in C5 and Eject to Waste.

9. Load tips. Mix Beads at D4. Transfer 100 µL of beads from D4 to Sample Plate at C4. Eject Tips.

10. Dispose of used Bead Plate (D4).

11. Move Sample Plate from C4 to D4. Load tips and Mix at D4.

12. Move lid from A2 to Sample Plate (D4).

13. Incubate 10 mins

During Incubation, Pre Broadcast ATL to 384-well Plate

a. Move lid A3 to B2. Move lid A4 to C4.

b. Load tips (single column).

c. Transfer 15 µL A-Tail Mix from Col 1 of Reagent Plate (A4) to Q2 of 384-well Plate (A3). Repeat until broadcast is complete. Eject Tips.

d. Move lid B2 to 384-well Plate (A3). Move lid C4 to Reagent Plate (A4).


15. Move Sample Plate (D4) to Magnet (B4). Incubate 5 min.

16. Load tips. Remove 90 µL supernatant from Sample Plate (B4) and dispense to liquid waste (A5). Repeat. Eject Tips.

17. Move lid C2 to A2.

18. Move lid from 80% EtOH (B5) to empty tip box (C5).

19. Load tips, wash beads with 150 µL 80% EtOH, remove EtOH to waste (A5), eject tips. Repeat.

20. Move lid from C5 back to B5.

21. Move beads (B4) to 37 ˚C (D2) to dry. Incubate 10 min.

22. Move beads (D2) to RT (D4).

23. Load tips, transfer 15 µL RSB (C2) to beads (D4), mix, incubate 3 min at RT.

24. Mix for 3 more cycles after shaking is complete. Eject tips.

25. Move lid A2 to RSB plate (C2).

Step 3: A-Tailing

1. Move lid from 384-well Plate (A3) to B2.

2. Load tips. Transfer 15 µL ATL from Q2 of 384-well plate (A3) to sample plate D4. Mix. Eject Tips.

3. Move lid from B2 to 384-well Plate (A3).

4. Incubate for 30 min at 37 ˚C at D4.

During Incubation, Pre-Broadcast Ligase Mix to 384-well Plate

a. Move lid A3 to B2. Move lid A4 to C4.


c. Transfer 8 µL Ligase Mix from Col 2 of Reagent Plate (A4) to Q4 of 384-well Plate (A3). Repeat until broadcast is complete. Eject Tips.





Step 4: Ligate Adapters


2. Load tips. Aspirate 3.5 µL of adapter from Q1 of 384-well plate (A3) and dispense 1 µL back to plate (A3). Dispense 2.5 µL to Sample Plate (D4).

3. Transfer 5 µL ligase mix (2 step aspirate) from Q4 of 384-well plate (A3) to Sample Plate (D4). Mix. Eject Tips.

4. Move lid C4 to Sample Plate (D4).

5. Incubate for 10 min at 30 ˚C

During Incubation, Pre Broadcast STL to 384-well Plate

a. Move lid A4 to C4.


c. Transfer 8 µL STL from Col 3 of Reagent Plate (A4) to Q3 of 384-well plate (A3). Repeat until broadcast is complete. Eject Tips.


6. Remove lid from Sample Plate (D4) to C4.


8. Load tips. Transfer 5 µL of STL (2 step aspirate) from Q3 of 384-well plate (A3) to Sample Plate (D4). Mix. Eject Tips.

9. Move lid B2 to 384-well Plate (A3)

10. Load tips. Transfer 12.5 µL PEG from B2 to Sample Plate (D4). Mix. Eject Tips.

11. Move lid C4 to Sample Plate (D4).

12. Incubate for 10 mins.

13. Remove lid from Sample Plate (D4) to C4.

14. Move Sample Plate (D4) to Magnet (B4)

15. Incubate for 10 mins.

During Incubation, Pre Broadcast PMM to PCR Plate

a. Load tips (single column).

b. Move Reagent Plate and Lid (A4) to D2.

c. Move top plate (of stack of 3) from D3 to A4.

d. Move lid from Reagent Plate (D2) to A2.

e. Transfer 30 µL from Col 11-12 of Reagent Plate (D2) to PCR Plate (A4). Repeat until broadcast is complete. Eject Tips.

f. Move lid A2 to Reagent Plate (D2).

g. Dispose of Lid from D2. Dispose of plate from D2.

16. Move Lid from C4 to PCR Plate (A4).

17. Load tips. Transfer 45 µL Supernatant from B4 to Liquid Waste (A5). Eject Tips.

18. Move lid from RSB plate (C2) to A2.

19. SPRI® cleanup (same as steps 18-21 under Step 2 End Repair).

20. Load tips, transfer 20 µL RSB (C2) to beads (D4), mix, incubate 5 min at RT.

21. Move Lid back to Resuspension Buffer Plate (C2) from A2.

Step 5: Setup PCR

1. Move Sample Plate (D4) to Magnet (B4). Incubate 5 mins.

2. Move lid from PCR Plate (A4) to C4.

3. Load tips. Transfer 20 µL from Sample Plate (B4) to PCR Plate (A4). Mix. Eject tips.

4. Move lid from C4 to PCR Plate (A4)

5. Move Head to A1. Show Application Complete Dialog.

Appendix B

Step-by-Step Guide to “TruSeq Post-PCR SPRI” Application

1. Load tips. Transfer 50 µL sample from PCR plate on magnet at B4 to clean PCR plate at D4. Eject tips.

2. Load tips. Mix AMPure® XP beads at C2. Transfer 50 µL beads from C2 to samples at D4. Mix.

3. Incubate beads and sample for 10 min. Dispose of bead plate at C2 and PCR plate at B4.

4. Move beads/sample from D4 to magnet at B4, incubate 10 mins.

5. Remove 100 µL supernatant to waste at A5. Eject tips.

6. Remove lid from 80% EtOH reservoir at B5 and place on empty tip box at C5.

7. Wash beads with 150 µL 80% EtOH two times. Replace lid on 80% EtOH reservoir.

8. Move beads to D2 to dry at 37 ˚C for 5 mins.

9. Move beads from D2 to D4.

10. Move lid from RSB at C4 to clean plate at C2.

11. Transfer 32 µL RSB from C4 to beads at D4. Mix. Eject tips.

12. Transfer sample/beads (D4) to magnet at B4, incubate 5 mins.

13. Move lid from C2 to C4. Move clean PCR plate from C2 to A4.

14. Transfer 30 µL sample from beads at B4 to clean plate at A4.

15. Move sample plate from A4 to D4 (4 ˚C). Move lid from C4 to D4.

16. Move head out of way and show application complete dialog.

For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs

Copyright ©2012, PerkinElmer, Inc. All rights reserved. PerkinElmer® is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 010526_01 Printed in USA Sep 2012

PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602www.perkinelmer.com


user’s guide illumina truseq dna - perkinelmer · user’s guide illumina truseq dna library...

Documents