Library Preparation with the LP 8.8.6 IFC Protocol (101 ...

101-8810 Rev 02PROTOCOL

Library Preparation with the LP 8.8.6 IFCFor use with the Advanta Solid Tumor and Advanta RNA Fusions NGS Library Prep Assays

Contents

About This Protocol 3

Safety Alert Conventions 3Safety Alerts for Chemicals 3Safety Alerts for Instruments 3

Safety Data Sheets 4

Introduction 4

Workflow Overview 5

Materials 5

Required Kits and Reagents 5Required Kits for the Solid Tumor Panel 5Required Kits for the RNA Fusions Panel 6Suggested Reagents from Other Suppliers 6Suggested Reagents for RNA Extraction 6Suggested Reagents for Positive Control 6Suggested Reagents for Quality Control 6

Consumables 7Required Consumables 7Suggested Consumables 7

Equipment 7Required Equipment 7Suggested Equipment 7

Required Software 8

Sample Requirements 8

Best Practices 8

Determine the Assay and Sample Plate Layouts 10

Prepare RT Reactions for the RNA Fusions Panel 11

Retrieve the Reagents 11

Prepare RT Reactions 11

Prepare the Assay Mixes and Sample Mixes 13


Prepare the Assay Mixes 13Prepare the Assay Pre-Mix 13Prepare the 10X Assay Mixes 14

Prepare Sample Mixes 16

Prepare the Sample Pre-Mix (DNA and/or RNA) 16Prepare the Sample Mixes 17

Prime the IFC 18

Load and Run the LP 8.8.6 IFC on Juno 19

Pool the Harvested Samples 22

Clean Up the Pooled Samples 23


Prepare the Reagents for Cleanup 24

First Cleanup (0.6X/1.2X Double-Size SPRI for Solid Tumor Panel, 0.6X/0.8X Double-Size SPRI for RNA Fusions Panel) 24

Second and Third Cleanups (0.8X SPRI) 26

Add the Sequencing Adapter to the Purified Library 28

Retrieve Reagents 28

Prepare Reagents 28

Clean Up the PCR Product (0.8X SPRI for Solid Tumor Panel, 0.6X SPRI for RNA Fusions Panel) 29

Perform Quality Control on the Sequencing Library (after sequencing adapter is added) 30

Sequence the Library 32

32

Appendix A: LP 8.8.6 IFC Bundled Kit Components 33

Appendix B: LP 8.8.6 Reagent Kit Components 33

Appendix C: Map of the LP 8.8.6 IFC 35

How to Use the Map 35

Appendix D: LP 8.8.6 IFC Scripts 36

Appendix E: LP 8.8.6 IFC One Step Thermal Protocol 36

Contents

Appendix F: Dual Indexing 37

Appendix G: TSP Library Barcode Plates 38

Plate Maps 38

Barcode Lists 40

Appendix H: Troubleshooting 44

Appendix I: Related Documents 45

Appendix J: Safety 45

General Safety 45

Instrument Safety 46

Chemical Safety 46

Disposal of Products 46

Library Preparation with the LP 8.8.6 IFC Protocol

2

About This Protocol

About This ProtocolThis protocol describes how to use the Advanta™ Solid Tumor NGS Library Prep Assay and the Advanta RNA Fusions NGS Library Prep Assay with the LP 8.8.6 integrated fluidic circuit (IFC) and the Juno™ system to perform library preparation for targeted sequencing on the Illumina® system. For detailed instructions on instrument and software operation, see the Juno System User Guide (100-7070).

IMPORTANT Before using this kit, read and understand the detailed instructions and safety guidelines in this document. For complete safety information, see Appendix J.

Safety Alert Conventions

Fluidigm documentation uses specific conventions for presenting information that may require your attention. Refer to the following safety alert conventions.

Safety Alerts for Chemicals

For hazards associated with chemicals, this document follows the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and uses indicators that include a pictogram and a signal word that indicates the severity level:

Safety Alerts for Instruments

For hazards associated with instruments, this document uses indicators that include a pictogram and signal words that indicate the severity level:

Indicator Description

Pictogram (see example) consisting of a symbol on a white background within a red diamond-shaped frame. Refer to the individual safety data sheet (SDS) for the applicable pictograms and hazards pertaining to the chemicals being used.

DANGER Signal word that indicates more severe hazards.

WARNING Signal word that indicates less severe hazards.

Indicator Description

Pictogram (see example) consisting of a symbol on a white background within a black triangle-shaped frame. Refer to the instrument user guide for the applicable pictograms and hazards pertaining to instrument usage.

DANGER Signal word that indicates an imminent hazard that will result in severe injury or death if not avoided.

WARNING Signal word that indicates a potentially hazardous situation that could result in serious injury or death if not avoided.

CAUTION Signal word that indicates a potentially hazardous situation that could result in minor or moderate personal injury if not avoided.

IMPORTANT Signal word that indicates information necessary for proper use of products or successful outcome of experiments.


3

Introduction

Safety Data Sheets

Read and understand the SDSs before handling chemicals. To obtain SDSs for chemicals ordered from Fluidigm, either alone or as part of this system, go to fluidigm.com/sds and search for the SDS using either the product name or the part number.

Some chemicals referred to in this protocol may not have been provided with your system. Obtain the SDSs for chemicals provided by other manufacturers from those manufacturers.

IntroductionLibrary preparation on the LP 8.8.6 IFC follows a two-step approach. During the first PCR, common sequence tags and sample barcodes are added to the target regions. The tags and barcode add ~100 bp to the target-specific amplicons. The second PCR adds another ~30 bp of Illumina adapter sequences to the amplicons. Therefore, the final library is 130 bp longer than the target-specific amplicons for single barcoding and 140 bp longer for dual barcoding.

The Advanta Solid Tumor NGS Library Prep Assay is optimized to enrich somatic mutation targets within 53 oncology-relevant genes utilizing DNA from solid tumor FFPE and fresh-frozen samples. The Advanta RNA Fusions NGS Library Prep Assay is optimized for fusion detection utilizing RNA from solid tumor FFPE samples. The LP 8.8.6 IFC contains six discrete partitions. Each partition processes up to eight samples against eight pools of assays. Using the LP 8.8.6 IFC and the Juno system, you can process samples with either the Solid Tumor Panel or RNA Fusions Panel individually, or both panels concurrently in the same run on the same IFC. For more information about planning your plate layouts in preparation for loading on the LP 8.8.6, see Determine the Assay and Sample Plate Layouts on page 10.

Figure 1. Overview of Advanta NGS assay chemistry

Tag 1SP

Target sequence

Tag 2

BC P7

P7

Harvest and pool(all sample amplicons in a single tube)

SPRI cleanup (3x)

SPRI cleanup (1x)

All steps occur in a single tube.

On-IFC barcoding

Tag 1 BCTag 2P5


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Workflow Overview

Workflow Overview

MaterialsRequired Kits and Reagents

For a list of the available kit bundles, see Appendix A. For a list of the reagent kit components, see Appendix B.

IMPORTANT Store reagents as soon as they are received, according to manufacturer’s storage recommendations.

Required Kits for the Solid Tumor Panel

Workflow Step Hands-On Time Run Time Estimated Time

Pre-PCR room

1 For RNA Fusions Panel only: Prepare reverse transcription (RT) reactions.

15 min 30 min 45 min

2 Prepare the 10X assay mixes and sample mixes. 40 min — 40 min

3 Prime the LP 8.8.6 IFC on Juno. 5 min 5 min 10 min

4 Load and run the LP 8.8.6 IFC on Juno and harvest samples.*

* Potential stopping point

10 min 4 hr 30 min 4 hr 40 min

Post-PCR room

5 Pool the harvested samples.* 10 min — 10 min

6 Clean up the pooled samples (3x).* 90 min — 90 min

7 Thermal-cycle the samples to add P5 sequencing adapters to the library.

10 min 60 min 70 min

8 Clean up the final sequencing library.* 20–30 min — 20–30 min

9 Quantify the sequencing library. 10–15 min — 10–15 min

Total 3 hr 30—45 min 6 hr 5 min 9 hr 35—50 min

Product Name Source Part Number Storage

Advanta Solid Tumor NGS Library Prep Assay—LP 8.8.6, 2 IFCs

Kit contains:

• Advanta Solid Tumor NGS Assay Pools

• Advanta NGS Library Prep Reagent Kit—LP 48.48 & LP 8.8.6, 2 IFCs

• Control Line Fluid LP 48.48 & LP 8.8.6

• Juno LP 48.48 & LP 8.8.6 Barrier Tape

• LP 8.8.6 IFCs

Fluidigm 101-7033

–20 °C

–20 °C

Room temperature

Room temperature

Room temperature

Targeted DNA Seq Barcode Plates*

* The entire set of four Targeted DNA Seq Barcode Plates supports 20 of the Advanta Solid Tumor NGS Library Prep Assay—LP 8.8.6, 2 IFCs kits.

Fluidigm 101-0744 –20 °C


5

Materials

Required Kits for the RNA Fusions Panel

Suggested Reagents from Other Suppliers

Suggested Reagents for RNA Extraction

Suggested Reagents for Positive Control

Suggested Reagents for Quality Control


Advanta RNA Fusions NGS Library Prep Assay—LP 8.8.6, 2 IFCs

Kit contains:

• Advanta RNA Fusions Reagent Kit

• Advanta NGS Library Prep Reagent Kit—LP 48.48 & LP 8.8.6, 2 IFCs

• Control Line Fluid LP 48.48 & LP 8.8.6

• Juno LP 48.48 & LP 8.8.6 Barrier Tape

• LP 8.8.6 IFCs

Fluidigm 101-8654

–20 °C

–20 °C

Room temperature

Room temperature

Room temperature

Targeted DNA Seq Barcode Plates* Fluidigm 101-0744 –20 °C

* The entire set of four Targeted DNA Seq Barcode Plates supports 20 of the Advanta RNA Fusions NGS Library Prep Assay—LP 8.8.6, 2 IFCs kits.

Product Name Source Part Number

Agencourt® AMPure® XP magnetic beads Beckman Coulter A63881 (60 mL)

DNase-free water Major laboratory supplier (MLS) —

Ethanol, absolute MLS —


Advanta FFPE RNA Extraction Kit:

• Advanta FFPE RNA Extraction Kit (50 reactions)

Fluidigm 101-6773

–20 °C

• Advanta FFPE Purification Beads Kit 4 °C


For Solid Tumor Panel:

• Tru-Q 2 (5% Tier) Reference Standard

• Tru-Q 3 (5% Tier) Reference Standard

Horizon Discovery

Horizon Discovery

HD729

HD730

For RNA Fusions Panel:

Seraseq® Fusion RNA Mix v3

SeraCare 0710-0431


Agilent® High Sensitivity DNA Kit Agilent Technologies 5067-4626

Qubit® dsDNA HS Assay Kit Thermo Fisher Scientific Q32851


6

Materials

Consumables

Required Consumables

Suggested Consumables

Equipment

Required Equipment

Suggested Equipment


Disposable microcentrifuge tubes, polypropylene, 1.5 mL MLS*

* Recommended: VWR® Slick Disposable Microcentrifuge Tubes, Polypropylene, 1.5 mL (VWR, PN 20170-666)

—

96-well PCR plates MLS†

† Recommended: TempPlate® semi-skirted 96-well PCR plates (USA Scientific, PN 1402-9700)

—

8-well PCR tube strips MLS —

Clear adhesive film for 96-well plates MLS‡

‡ Recommended: MicroAmp® Clear Adhesive Film (Thermo Fisher Scientific, PN 4306311)

—

5 mL or 15 mL graduated tubes MLS —


Qubit Assay Tubes Thermo Fisher Scientific

Q32856

1.5 mL screw cap tubes MLS —


Juno Fluidigm 101-6455

2100 Bioanalyzer® Agilent G2940CA

DynaMag™-2 Magnet or equivalent Thermo Fisher Scientific

12321D

Pipettes (P2–P1000) and appropriate low-retention tips*

* Rainin® pipettes recommended.

MLS —

8-channel pipettes and appropriate low-retention tips* MLS —

Vortexer MLS —

Microcentrifuge MLS —

Centrifuge with rotor to accommodate 96-well plates MLS —

Standard thermal cycler MLS —


Qubit fluorometer Thermo Fisher Scientific

—

DNA hood and DNA-free hood MLS —

Heating block (37 °C) MLS —


7

Sample Requirements

Required Software

Juno Software v3.13.1 or later is required for this protocol. For software updates, go to fluidigm.com/software.

Sample RequirementsFor better traceability, assemble your samples in a 96-well PCR plate and record in a sample map.

IMPORTANT Multiple panels can be processed on the same IFC. However, libraries for each panel must be harvested separately and normalized before combining and sequencing. Failure to independently harvest and normalize the libraries results in unequal sequencing read distributions between panels.

For the Solid Tumor Panel

This protocol supports use of 12.5 ng of human genomic DNA (gDNA) that is at least 200 bp in a maximum of 2.5 μL. However, adding increased amounts of total DNA results in higher-quality libraries, particularly for DNA of lower or unknown quality. We recommend using DNA samples with an A260/A280 ratio that is >1.5.

For the RNA Fusions Panel

The RNA samples must be DNA-free. A range of 10–100 ng of sample-specific total RNA is recommended for each reverse transcription (RT) reaction. Success of a given sample for relative quantitation using qPCR depends on both the RNA sample quality and the abundance of expressed RNA transcripts within the sample. Adding higher amounts of total RNA within this range results in higher-quality libraries, particularly for RNA of lower or unknown quality. For high quality samples we recommend using no more than 10 ng of total RNA input. Positive control sample input should be adjusted so that the reads assigned to the positive control sample are no more than 10% of the total number of reads assigned to all RNA fusion samples on the IFC.

NOTE Using samples outside of the recommended concentration range may increase variation in the results.

Best PracticesIMPORTANT Read and understand the safety information in Appendix J.

For the overall success of the protocol, we recommend the following best practices.

IFC and Control Line Fluid Handling

• Use the IFC within 24 hr of opening the package.

• Inspect the IFC for any signs of visible damage before use. Ensure that the barcode label is intact and the IFC surfaces are clear of particulates.


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Best Practices

• Do not evacuate air from syringes prior to injecting Control Line Fluid.

• Avoid bending the syringe tip.

• Be careful when removing the syringe cap to prevent drips.

• Before removing the syringe from the accumulator, ensure that all of the Control Line Fluid and air are purged from the syringe to avoid dripping fluid on the surface of the IFC.

• Avoid getting Control Line Fluid on the exterior of the IFC or in the inlets because this makes the IFC unusable. If this occurs, use a new IFC.

• During use, take care to avoid the introduction of particulates, reagents, and fluids to the surface of the IFC.

Reagent Handling

• Use good laboratory practices to minimize contamination of samples:

• Use a new pipette tip for every new sample.

• Whenever possible, separate pre- and post-PCR activities. Dedicate laboratory materials to designated areas.

• Ensure that lab consumables (tubes, tips, plates) used for the RNA handling steps are RNase-free.

• Retrieve only the reagents required from each kit based on the number of IFCs that you will run.

• Use only the reagents provided in the required kit.

• Do not swap reagents between kit lots.

• Unless otherwise specified, thaw reagents at room temperature (15–30 °C), and then use them at room temperature.

• Mix and centrifuge reagents as directed.

• Place the sample mixes on ice when not in use.

• To reduce the number of pipetting steps, we recommend first transferring reagents into an 8-well PCR tube strip to enable transfer into a 96-well plate using an 8-channel pipette.

Bubble Prevention

• Avoid creating bubbles while vortexing reagents and when transferring reagents to the IFC.

• Vortex gently (low speed) but thoroughly (at least 5 sec) to ensure that all reagents and reagent mixes are homogeneous.

• After vortexing, centrifuge to collect all mixes at the bottoms of the wells before pipetting into the IFC inlets. Failure to do so may result in a decrease in data quality.

• Check the source plate or tube for bubbles before pipetting.

• Check pipette tips for air gaps while pipetting.

• Pipet reagents slowly and carefully to transfer entire volumes and to minimize bubbles.


9

Best Practices

• To avoid creating bubbles in the IFC inlets, pipet into the inlets at an angle and do not go past the first stop on the pipette. If a bubble is introduced, ensure that it floats to the top of the inlet.

• If necessary, remove bubbles from an IFC inlet by removing the contents of the inlet by pipette and then carefully re-pipetting the contents into the inlet.

Determine the Assay and Sample Plate Layouts

The LP 8.8.6 IFC contains six independent partitions, each of which can run a different assay panel. When setting up your assay and sample plate layouts:

• Lay out your assay and sample plates in columns. Each column will fill one partition.

• Group assays by panel type. Use only the assays from a single panel in a partition. Do not combine panels within a partition.

• Put DNA samples in different partitions than RNA (cDNA) samples.

• Group similar samples in the same column/partition.

• Group samples of similar quality in the same column/partition.

6

6

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6

6

6

6

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6

6

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5

5

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5

5

5

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5

5

4

4

4

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4

4

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4

4

4

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4

4

4

4

4

3

3

3

3

3

3

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3

3

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3

3

2

2

2

2

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2

2

2

2

2

2

2

2

2

2

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1A

1B

1C

1D

1E

1F

1G

1H

2A

2B

2C

2D

2E

2F

2G

2H

3A

3B

3C

3D

3E

3F

3G

3H

4A

4B

4C

4D

4E

4F

4G

4H

5A

5B

5C

5D

5E

5F

5G

5H

6A

6B

6C

6D

6E

6F

6G

6H

96-well plate for assay or sample mixes LP 8.8.6 IFC

Sample inlets Assay inlets

For partition 1

For partition 2

For partition 3

For partition 4

For partition 5

For partition 6


10

Prepare RT Reactions for the RNA Fusions Panel

Prepare RT Reactions for the RNA Fusions PanelIf you are using the Solid Tumor Panel, skip this section and go to Prepare the Assay Mixes and Sample Mixes on page 13.

Retrieve the Reagents

Prepare RT Reactions

1 Manually flick the bottom of TSP Reverse Transcriptase and RNase Inhibitor tubes with your forefinger to mix the enzymes (do not vortex). Vortex all other reagents for 20 sec, and then briefly centrifuge all reagents before use.

2 On ice, prepare the RT pre-mix in a 1.5 mL PCR tube as shown in Table 1.

Table 1. RT pre-mix

3 Vortex the sample pre-mix for 10–20 sec at a medium speed, and then briefly centrifuge to bring down all components and remove bubbles.

4 On ice, prepare the RT sample mix in a 96-well PCR plate as shown in Table 2.

NOTE If you are running more than 8 samples, we recommend first transferring equal amounts of the RT pre-mix into each well of an 8-well strip, and then using an 8-channel pipette to transfer the RT pre-mix from the strip into the 96-well plate.

Required Reagent Preparation

Total RNA

Remove from –20 °C, thaw, and keep on ice.TSP Reverse Transcriptase (Fluidigm PN 101-8818)

TSP RNase Inhibitor (Fluidigm PN 101-8911)

PCR Water (Fluidigm PN 100-5941)Remove from –20 °C, thaw to room temperature.

5X TSP RT Buffer (Fluidigm PN 101-8913)

Component Volume perReaction

(μL)

Volume for8 Reactions/

1 Partition (μL)*†

* Includes overage for ease of pipetting

† Each partition of the LP 8.8.6 IFC contains 8 reactions. Scale up pre-mix appropriately for the number of times you are running the RNA Fusions Panel.

Volume for6 Partitions/

1 IFC (μL)*

PCR Water (Fluidigm PN 100-5941) 0.65 6.5 39

5X TSP RT Buffer (Fluidigm PN 101-8913) 1 10 60

TSP Reverse Transcriptase(Fluidigm PN-101-8818)

0.25 2.5 15

TSP RNase Inhibitor (Fluidigm PN 101-8911)

0.1 1 6

Total 2 20 120


11

Prepare RT Reactions for the RNA Fusions Panel

Table 2. RT sample mix

5 Properly seal the plate with a PCR-compatible adhesive film and gently vortex to mix the RT reactions.

6 Centrifuge the RT reaction plate at 2,500–3,000 x g for 1 min and then place them in a standard thermal cycler.

7 Incubate the RT reactions using the following thermal protocol:

STOPPING POINT The reverse transcription reaction products (cDNA) can be used to prepare the sample mixes for IFC loading or stored at −20 °C for later use.

8 While the RT reaction is running, prepare the assays and samples. The reverse transcription reaction takes approximately 30 min.

Component Volume per Reaction (μL)

RT pre-mix (see Table 1) 2.0

Total RNA, 10–100 ng 3.0

Total 5.0

Temperature Time

25 °C 10 min

50 °C 10 min

85 °C 5 min

4 °C ∞


12

Prepare the Assay Mixes and Sample Mixes

Prepare the Assay Mixes and Sample MixesPrepare the assay and sample mixes in a pre-PCR room.

IMPORTANT Pipet reagents slowly and carefully to transfer entire volumes and to minimize bubbles. Reagents tend to cling to tip surfaces and can form bubbles easily.


Prepare the Assay Mixes

When preparing the assay mixes in the 96-well plate, follow the best practices for determining assay plate layouts on page 10.

Prepare the Assay Pre-Mix

This protocol prepares sufficient assay pre-mix for 48 reactions plus overage to compensate for dead volume and to allow for proper pipetting volumes.

1 Vortex reagents for 20 sec, and then briefly centrifuge them before use.

2 In a DNA-free hood, combine the components shown in Table 3 in a new 1.5 mL microcentrifuge tube.


Assay Pools

• Solid Tumor Panel (Fluidigm PN 101-8816)

and/or

• RNA Fusions Panel (Fluidigm PN 101-8817)

NOTE You can use assays from both the Solid Tumor and RNA Fusions Panels in the same IFC. If frozen, thaw to room temperature.

Targeted DNA Seq Barcode Plates (Fluidigm PN 101-0744)

Samples

• For Solid Tumor Panel: genomic DNA sample (12.5 in 2.5 μL)

• For RNA Fusions Panel: cDNA (see Prepare RT Reactions for the RNA Fusions Panel on page 11)

TSP Sample Loading Reagent v2 (Fluidigm PN 101-7634)

Remove from –20 °C and thaw to room temperature.

PCR Water (Fluidigm PN 100-5941)

TSP Harvest Reagent (Fluidigm PN 101-0743)

NOTE Due to the larger volume, this reagent takes longer to thaw than the other reagents.

TSP Assay Loading Reagent (Fluidigm PN 101-0409)

4X TSP Master Mix (Fluidigm PN 101-3055)

TSP DNA Polymerase (Fluidigm PN 101-0995) Remove from –20 °C and keep on ice.

DNA Suspension Buffer (Teknova PN T0221) Remove from storage and keep at room temperature.


13


Table 3. Assay pre-mix

3 Gently vortex the assay pre-mix for 5 sec at medium speed, and then use a microcentrifuge for ≥3 sec to bring down all components and remove bubbles.

Prepare the 10X Assay Mixes

Prepare the 10X assay mixes in a 96-well plate.

IMPORTANT If you are running multiple panels on the same IFC, each panel type must be in a separate partition. Do not mix panels within a partition.

Figure 2. Layout of 10X assay mixes (per-well transfer volumes)

1 Immediately before use, ensure that each tube of Assay Pools is securely sealed, and then vortex at medium-high speed for 10–20 sec to mix. Centrifuge the assay pools tubes at 3,000 × g for 5 min.

Component Volume for1 Partition/8 Assay

Inlets (μL)*

* Includes overage

Volume for1 IFC/48 Assay

Inlets (μL)*

PCR Water (Fluidigm PN 100-5941) 33 198

TSP Assay Loading Reagent (Fluidigm PN 101-0409)

3 18

Total 36 216

1A

1B

1C

1D

1E

1F

1G

1H

2A

2B

2C

2D

2E

2F

2G

2H

3A

3B

3C

3D

3E

3F

3G

3H

4A

4B

4C

4D

4E

4F

4G

4H

5A

5B

5C

5D

5E

5F

5G

5H

6A

6B

6C

6D

6E

6F

6G

6H

2.50 μL Assay Pools/well OR 2.50 μL PCR Water/well(if well does not contain Assay Pools)

3.75 μL assay

pre-mix/well

Assay mixes in a 96-well plate

Assays for partition 1







14


2 In a DNA-free hood, prepare an assay mix for each of the 8 tubes of Assay Pools in a new 96-well plate by adding each component as shown in Table 4 and Figure 3. Pipet gently up and down to mix, being careful to avoid creating bubbles.

Table 4. Assay mixes

Figure 3. Preparation of assay mixes

3 Seal the plate, and then centrifuge at 2,500–3,000 x g for 1 min to bring down contents. Centrifuge longer if any bubbles are present. Label the plate and set aside until you are ready to load the IFC.

Component Volume perAssay Pool/Well (μL)*

* Includes overage

Assay pre-mix (see Table 3) 3.75

Assay Pools or PCR Water†

† For unused assay inlets, replace the diluted Assay Pools with 2.5 μL of PCR Water (Fluidigm PN 100-5941).

2.50

Total 6.25

AdvantaNGS Assay Pools

Assay mixes in96-well plate

Assay pre-mix

3.75 μL/well 2.5 μL/well1A

1B

1C

1D

1E

1F

1G

1H

2A

2B

2C

2D

2E

2F

2G

2H

3A

3B

3C

3D

3E

3F

3G

3H

4A

4B

4C

4D

4E

4F

4G

4H

5A

5B

5C

5D

5E

5F

5G

5H

6A

6B

6C

6D

6E

6F

6G

6H 8

7

6

5

4

3

2

1


15


Prepare Sample Mixes

When preparing the sample mixes in the 96-well plate, follow the best practices for determining sample plate layouts on page 10.

IMPORTANT All 48 sample inlets of the IFC must be filled with reagent for proper loading.

Prepare the Sample Pre-Mix (DNA and/or RNA)

Prepare sufficient volumes of the DNA and/or RNA sample pre-mixes for the number of partitions you are using for each panel. Refer to Table 5 to calculate the volumes required for the desired number of partitions.

This protocol prepares the sample pre-mix with enough overage to compensate for dead volume and to allow for proper pipetting volumes.

1 Manually flick the bottom of TSP DNA Polymerase tube with your forefinger to mix the enzyme (do not vortex). Vortex all other reagents for 20 sec, and then briefly centrifuge all reagents before use.

2 Label two new 1.5 mL tubes, one for each sample pre-mix (DNA or RNA).

3 In a DNA-free hood, prepare the sample pre-mix in a new 1.5 mL microcentrifuge tube using volumes shown in Table 5.

IMPORTANT• Add 4X TSP Master Mix to PCR Water to dilute it before adding the remaining reagents. • While pipetting, do not go past the first stop on the pipette.• 4X TSP Master Mix is viscous. Pipet slowly.

Table 5. Sample pre-mix. Combine and mix in the order shown.

4 Vortex the sample pre-mix for 10–20 sec at a medium speed, and then use a microcentrifuge for 10 sec to bring down all components and remove bubbles.

5 Pipet 19 μL of the sample pre-mix into each well of a new 8-well strip (see Figure 4 on page 17).

IMPORTANT To prevent introducing bubbles, pipet only to the first stop during this transfer process. To help ensure that all liquid can be retrieved when preparing the sample mixes, we recommend using a microcentrifuge at maximum speed for 3 sec.

Component Volume perpartition (μL)*

* Includes overage

Volume perIFC (μL)*

1 PCR Water (Fluidigm PN 100-5941) 6.7 40

2 4X TSP Master Mix (Fluidigm PN 101-3055)

16.7 100

3 TSP Sample Loading Reagent v2 (Fluidigm PN 101-7634)

3.3 20

4 TSP DNA Polymerase (Fluidigm PN 101-0995, do not vortex)

2.7 16

Total 29.4 176


16


Prepare the Sample Mixes

Figure 4. Preparation of sample mixes (per-well transfer volumes)

1 Centrifuge the TSP Barcode Plate at 3,000 × g for 3 min before using it.

2 In a DNA sample hood, prepare the sample mixes by pipetting the components shown in Table 6 into each well of a new 96-well plate. Pipet reagents and samples according to the diagram shown in Figure 4. Use an 8-channel pipette to transfer the sample pre-mix from the 8-well strip.

3 Pipet PCR Water into the wells that do not contain samples.

Table 6. Sample mixes

NOTE For a complete list of barcodes and barcode maps, see Appendix G.

4 Reseal the Targeted DNA Seq Barcode Plate. If using the barcode plate again within 2 days, store at 4 °C. Otherwise, store at −20 °C.

5 Tightly seal the 96-well plate with clear adhesive film, vortex thoroughly for 20 sec, and then centrifuge the plates at 2,500–3,000 × g for 5 min.

Samplepre-mix in 8-well strip

96-well plate

2.5 μL sample/well and 1.25 μL barcode primer/well

3.75 μL PCR Water/well(if well does not contain samples)

2.5 μL/well

OR

1A

1B

1C

1D

1E

1F

1G

1H

2A

2B

2C

2D

2E

2F

2G

2H

3A

3B

3C

3D

3E

3F

3G

3H

4A

4B

4C

4D

4E

4F

4G

4H

5A

5B

5C

5D

5E

5F

5G

5H

6A

6B

6C

6D

6E

6F

6G

6H

Samples for partition 1






Component Volume perReaction (μL)*

* Includes overage

Volume per Reaction for InletsThat Do Not Contain Samples (μL)*

Sample pre-mix (see Table 5) 2.5 2.5

Sample

• For Solid Tumor Panel: Genomic DNA sample (12.5 ng in 2.5 μL)†

• For RNA Fusions Panel: cDNA (see page 11)

† Adding higher amounts of DNA within this range results in higher-quality libraries, particularly for DNA of lower or unknown quality.

2.5 —

Barcode primer from TSP Barcode Plate (Fluidigm PN 101-0744)

1.25 —

PCR Water (Fluidigm PN 100-5941) — 3.75

Total 6.25 6.25


17


6 If you observe bubbles in the wells following centrifugation, manually flick or gently snap the bottom of the affected wells with your forefinger, and then centrifuge the plates again at 2,500–3,000 × g for 5 min.

7 Continue to Prime the IFC.

Prime the IFC

To aid pipetting, print the actual-size map of the LP 8.8.6 IFC in Appendix C and place it under the IFC.

For detailed instructions about injecting Control Line Fluid, see the Control Line Fluid Loading Procedure (68000132). For detailed instructions about using Juno, see the Juno System User Guide (100-7070). For a description of the Juno scripts, see Appendix D.

IMPORTANT When injecting Control Line Fluid:• Follow the best practices for handling IFCs and Control Line Fluid on page 8.• Use only LP 48.48 & LP 8.8.6 syringes containing 300 μL of Control Line Fluid.

1 Turn on the Juno instrument, if necessary. Initialization takes approximately 5 min.

2 Ensure that the MX Interface Plate is installed in the instrument.

3 Pull the protective tape down and away from the bottom of the IFC.

4 Inject Control Line Fluid into each accumulator on the IFC (see Figure 5). Holding the IFC at a 45° angle, insert the syringe tip into one of the spaces between the arms of the “X” at the top of the valve and press down gently to move the black O-ring to the side (see Figure 6 on page 19).

Figure 5. LP 8.8.6 IFC priming map

H3

H1 H2

H4

AccumulatorAccumulator

Harvest reservoirs

Harvest reservoirs

TSP Harvest Reagent, 650 μL

LP 8.8.6 Control Line Fluid, 300 μL


18

Load and Run the LP 8.8.6 IFC on Juno

Figure 6. Injecting Control Line Fluid into the accumulators on the LP 8.8.6 IFC

5 Place the IFC on a flat surface and pipet 650 μL of TSP Harvest Reagent into each of the harvest reservoirs H1, H2, H3, and H4 (see Figure 5 on page 18).

6 On Juno, tap OPEN to open the instrument tray. Place the IFC on the tray and align the notched corner of the IFC to the white notch on the tray. Tap LOAD.

7 On the Scripts screen, tap Prime LP–8.8.6 and tap Run.

IMPORTANT Load the IFC within 60 min of completing the Prime script.

Load and Run the LP 8.8.6 IFC on JunoIMPORTANT When loading the assay and sample mixes in the IFC:• Avoid creating bubbles (see Best Practices on page 8).• Before pipetting reagents, maintain traceability by noting the orientation of the A1 corner,

assay inlets, and sample inlets, as shown in Step 4 on page 20.

1 Immediately before use, ensure that the assay and sample mix plates are securely sealed, and then vortex thoroughly for 20 sec to mix. Centrifuge the plates at 2,500–3,000 × g for 5 min.

2 If you observe bubbles in the wells following centrifugation, manually flick or gently snap the bottom of the affected wells with your forefinger, and then centrifuge the plates again at 2,500–3,000 × g for 5 min.

3 After the Prime script is finished on Juno, tap EJECT to eject the IFC.

45°

Syringe tip inserted into one of the spaces between the arms of the “X”

Move the O-ring to the side.


19


4 Pipet the assay mixes and sample mixes into the LP 8.8.6 IFC, as shown.

a Carefully pipet 4.0 μL of each assay mix from the plate into the designated assay inlets on the IFC.

b Carefully pipet 4.0 μL of each sample mix from the plate into the designated sample inlets of the IFC based on the predefined sample map.

96-well platecontaining

assay mixes

4 μL

Assay inlets

1A

1B

1C

1D

1E

1F

1G

1H

2A

2B

2C

2D

2E

2F

2G

2H

3A

3B

3C

3D

3E

3F

3G

3H

4A

4B

4C

4D

4E

4F

4G

4H

5A

5B

5C

5D

5E

5F

5G

5H

6A

6B

6C

6D

6E

6F

6G

6H

7 8

19 20

9 10

21 22

11 12

23 24

31 32

43 44

55 56

33 34

45 46

57 58

35 36

47 48

59 60

67 68

79 80

69 70

81 82

71 72

83 84

91 92 93 94 95 96

6

6

6

6

6

6

6

6

5

5

5

5

5

5

5

5

4

4

4

4

4

4

4

4

3

3

3

3

3

3

3

3

2

2

2

2

2

2

2

2

1

1

1

1

1

1

1

1

Assay inlets for partition 1






96-well platecontainingsample mixes

4 μL

Sample inlets

1A

1B

1C

1D

1E

1F

1G

1H

2A

2B

2C

2D

2E

2F

2G

2H

3A

3B

3C

3D

3E

3F

3G

3H

4A

4B

4C

4D

4E

4F

4G

4H

5A

5B

5C

5D

5E

5F

5G

5H

6A

6B

6C

6D

6E

6F

6G

6H

7 8

19 20

9 10

21 22

11 12

23 24

31 32

43 44

55 56

33 34

45 46

57 58

35 36

47 48

59 60

67 68

79 80

69 70

81 82

71 72

83 84

91 92 93 94 95 96

6

6

6

6

6

6

6

6

5

5

5

5

5

5

5

5

4

4

4

4

4

4

4

4

3

3

3

3

3

3

3

3

2

2

2

2

2

2

2

2

1

1

1

1

1

1

1

1

Sample inlets for partition 1







20


5 Cover the sample and assay inlets with LP 48.48 & LP 8.8.6 Barrier Tape:

a Gently pull the tab on the adhesive side of one barrier tape to expose the adhesive.

b With the adhesive side facing the IFC, insert one end of the tape into the top edge of the sample chamber, and then gently drop in the barrier tape so that it covers the chamber (Figure 7A).

c Repeat Steps a–b to cover the assay chamber of the IFC.

d Ensure that both barrier tapes are seated flat in the chambers.

e Insert the two diagonal posts of the Barrier Tape Applicator through the holes of the Barrier Tape Applicator Adapter. The flat side of the adapter is against the applicator. The applicator and the adapter magnetically attach (Figure 7B).

f Flip the applicator-adapter assembly over so that the adapter faces the IFC.

g With the IFC on the laboratory bench, align the applicator-adapter assembly with the IFC and then gently push the assembly down on the IFC for 1–2 sec. Gently lift the assembly from the IFC. The barrier tapes are properly adhered to the IFC chambers (Figure 7C).

Figure 7. Covering inlets with barrier tape

h To release the adapter from the applicator, hold the applicator-adapter assembly just above the laboratory bench and press down the left and right central edges of the central ejector. The adapter will drop off the applicator.

i Store the Barrier Tape Applicator and Barrier Tape Applicator Adapter for next use.

7A. Insert the barrier tape with adhesive side down into the sample and assay chambers:

7B. Insert the two diagonal posts of the applicator through the holes of the adapter:

7C. Push down on the applicator so that the tape adheres to the IFC:

Sample chamber

Assay chamber

Left, right edges of ejector


21

Pool the Harvested Samples

6 On Juno, tap OPEN and place the IFC on the tray of the Juno instrument, and then tap LOAD.

7 On the Scripts screen, tap One Step LP–8.8.6.

8 Select when the script should finish (if necessary, adjust the harvest time), and then tap RUN. The run takes approximately 4 hr to complete the load/mix, thermal cycle, and harvest steps. You can delay the harvest step for up to 16 hr.

NOTE For a description of the One Step LP–8.8.6 thermal protocol, see Appendix E.

9 After the run is finished, tap EJECT to eject the IFC.

IMPORTANT Eject the IFC ≤60 min after the run is complete.

10 After ejecting the IFC, immediately proceed to the next section.

NOTE The volume of TSP Harvest Reagent in H1 will be lower than in the other three harvest reservoirs.

Pool the Harvested SamplesPool the harvested samples in a post-PCR room. Each processed sample is harvested from the same sample inlet that was used to dispense sample mix into the IFC. Pool the harvested samples by first transferring the desired samples from the IFC to an 8-well strip, and then transferring them to a 1.5 mL tube, as shown in Figure 8.

IMPORTANT Pool the harvested samples by the panel type used to allow for independent normalization of each panel to achieve the desired sequencing depth. Normalized final libraries can then be combined and sequenced together, if desired. If you ran multiple panels on the same IFC, harvest the barcoded amplicons from each panel type to a separate tube.

Any unused partitions do not need to be pooled.

Figure 8. Process for pooling harvested samples

1 Carefully remove the LP 48.48 & LP 8.8.6 Barrier Tape from the sample chamber of the IFC by placing the IFC on a flat surface, holding the IFC with one hand, and slowly pulling the tab of the barrier tape until the tape is peeled away from the sample inlets.

24

21

18

15

12

9

6

3

42

39

36

33

30

27

48

45

22

19

16

13

10

7

4

1

23

20

17

14

11

8

5

2

44

41

38

35

32

29

26

4746

43

40

37

34

31

28

25

LP 8.8.6 IFC 8-well strip 1.5 mL tube

DO NOT USESample inlets containingharvested samples


22

Clean Up the Pooled Samples

2 Set an 8-channel pipette to 12.0 μL to transfer and combine the entire harvest volumes from the sample inlets of the LP 8.8.6 IFC directly into an 8-well strip. Each harvest volume should be >5 μL.

IMPORTANT Be sure to transfer the entire volume from each sample inlet for best barcode uniformity of mapped reads.

NOTE Because all samples are barcoded, it is not necessary to change pipette tips when harvesting and pooling the samples.

3 Combine entire harvest volumes from the samples directly into an 8-well strip.

4 Combine volumes from the 8-well strip into a single new 1.5 mL microcentrifuge tube.

NOTE If you are processing more than one sample pool at the same time for cleanup, label a new 1.5 mL tube for each sample pool to be processed.

STOPPING POINT Store the 1.5 mL tube(s) of pooled samples at 4 °C for up to one week or at –20 °C for longer storage.

5 If continuing immediately to cleaning up the pooled samples, retrieve the Agencourt AMPure XP magnetic beads from storage now and warm them to room temperature for 30 min.

Clean Up the Pooled SamplesClean up the pooled samples in a post-PCR room. Perform three bead-based cleanups before adding the sequencing adapter, followed by one bead-based cleanup after PCR.

Process the harvested samples pooled from each panel and IFC independently for the cleanup and PCR steps.

IMPORTANT• To minimize sample cross-contamination, it is critical to remove all excess primers from the

pooled samples before adapter addition. Due to the high concentration of primers remaining in the harvest product, three sequential solid-phase reversible immobilization (SPRI) bead cleanup steps are required. Pipet carefully to ensure proper SPRI (bead:DNA) ratios.

• Fully dispense the magnetic bead suspension from the pipette tip.



DNA Dilution Reagent (Fluidigm PN 100-9167) Remove from –20 °C and thaw to room temperature.

Agencourt AMPure XP magnetic beads(Beckman Coulter PN A63880)

Remove from storage and keep at room temperature.

DNase-free water

Ethanol, absolute


23


Prepare the Reagents for Cleanup

A 5 mL preparation of 80% ethanol is sufficient for three cleanups of a single pool of harvested samples and the final cleanup of the sequencing library. Scale the preparation of 80% ethanol as necessary to process all IFC harvested sample pools that might be prepared in parallel.

IMPORTANT Ethanol is hygroscopic. Prepare fresh 80% ethanol before library cleanup. Cap the tube of 80% ethanol when not in use. A batch of ethanol can be kept for 24 hr.

1 Remove the Agencourt AMPure XP magnetic beads from 4 °C, and then warm the beads to room temperature for 30 min before use.

2 Vortex the Agencourt AMPure XP magnetic beads vigorously to ensure that they are fully suspended.

3 Using a new graduated tube, prepare 5 mL of fresh 80% ethanol for each pool of harvested samples:

a Pipet 1 mL of DNase-free water into the tube.

b Add 4 mL of absolute alcohol to bring the volume to 5 mL.

c Cap the tube, and then invert to mix.

First Cleanup (0.6X/1.2X Double-Size SPRI for Solid Tumor Panel, 0.6X/0.8X Double-Size SPRI for RNA Fusions Panel)

The first of three cleanups is a double-size SPRI step where the ratio of beads to sample removes large DNA fragments first, then removes the smaller primer fragments.

IMPORTANT In Step 5 of this section, do not discard the supernatant.

NOTE If you are processing more than one sample pool at the same time, label a new 1.5 mL tube for each sample pool to be processed.

1 Suspend magnetic beads in pooled samples:

a Pipet pooled samples into a new 1.5 mL microcentrifuge tube. If the volume of pooled samples is <150 μL, add DNA Dilution Reagent or PCR Water to bring the volume to 150 μL. Label and store the remaining pooled samples for possible contingencies.

b Vortex the Agencourt AMPure XP magnetic beads at high speed for 1 min. The beads should appear homogeneous and uniform in color.

c Pipet Agencourt AMPure XP magnetic beads into each tube that contains pooled samples. Expel any beads left in the pipette tip by pipetting the suspension up and down 10 times.

Component Volume forFirst Cleanup (μL)

Pooled samples 150


90

Total 240


24


2 Vortex the suspension at high speed for 20 sec and centrifuge the tubes briefly for a few seconds to bring the liquid to the bottom.

3 Incubate the suspension at room temperature for 10 min.

4 Briefly centrifuge the tube, and then place the tube on a magnetic stand for 1–2 min until the solution is clear.

5 Without disturbing the beads, and keeping the tube on the magnetic stand, carefully pipet the supernatant to a new tube.

IMPORTANT Retain all of the supernatant.

Figure 9. Transfer the supernatant into a new tube.

Use a P10 pipette to transfer any residual volume to ensure that all supernatant has been transferred.

6 Dispose of the tube containing the beads.

7 Vortex the bottle of AMPure XP magnetic beads at high speed for 20 sec, and then add beads into the supernatant:

8 Vortex the suspension at high speed for 20 sec and centrifuge the tubes briefly for a few seconds to bring the liquid to the bottom.



11 Without disturbing the beads, and keeping the tube on the magnetic stand, remove and discard the supernatant.

12 Use a P10 pipette to remove any residual supernatant from the tube.

13 Wash the beads 3 times with 80% ethanol:

a Keeping the tube on the magnetic stand, pipet 400 μL of 80% ethanol to wash the beads.

into a new 1.5 mL tube

Transfersupernatant

Beads(Discard after removing supernatant.)

DNA Libraries RNA Libraries

Component Volume forSolid Tumor Panel (μL)

Volume forRNA Fusions Panel (μL)

Supernatant Entire volumefrom Step 5

Entire volumefrom Step 5

Agencourt AMPure XP magnetic beads 90 30

Total 330 270


25


b Incubate the tube at room temperature for 30–60 sec.

c Without disturbing the beads, and keeping the tube on the magnetic stand, remove and discard the ethanol.

d Repeat Steps a–c 2 more times for a total of 3 washes. Completely remove and discard all of the 80% ethanol.

14 Transfer the tube to a rack and open the tube. Remove any remaining ethanol by drying the beads at 37 °C for 1 min or air-drying the beads at room temperature for 10–15 min.

15 Prepare the eluate:

a To the dried beads, pipet 30 μL of DNA Dilution Reagent. Vortex the suspension at high speed for 20 sec.

b Incubate the suspension at room temperature for 2 min.

c Briefly centrifuge the tube, and then place the tube on a magnetic stand for 1–2 min until the solution is clear.

d Keeping the tube on the magnetic stand, pipet 30 μL of the eluate to a new tube.

STOPPING POINT You can store the eluate at 4 °C for up to one week or at –20 °C for longer storage.

Second and Third Cleanups (0.8X SPRI)

1 Suspend magnetic beads in eluate:

a Vortex the Agencourt AMPure XP magnetic beads at high speed for 20 sec. The beads should appear homogeneous and uniform in color.

b Pipet Agencourt AMPure XP magnetic beads into the same tube with the eluate from the previous cleanup. Expel any beads left in the pipette tip by pipetting the suspension up and down 10 times.

2 Vortex the suspension at high speed for 20 sec.





Component Volume for Second andThird Cleanup (μL)

Eluate 30


24

Total 54


26







8 Transfer the tube to a rack and open the tube. Remove any remaining ethanol by drying the beads at 37 °C for 1 min or air-drying the beads at room temperature for 10–15 min.

9 Prepare the eluate:

a To the dried beads, pipet 30 μL of DNA Dilution Reagent. Vortex the suspension at high speed for 20 sec.

b Incubate the suspension at room temperature for 2 min.

c Briefly centrifuge the tube, and then place the tube on a magnetic stand for 1–2 min until the solution is clear.

d Keeping the tube on the magnetic stand, pipet 30 μL of the eluate to a new tube.

STOPPING POINT You can store the eluate from the second cleanup at 4 °C for up to one week or at –20 °C for longer storage.

10 Perform the third cleanup (0.8X SPRI) by repeating Steps 1–9 with 30 μL of the eluate from the second cleanup. The eluate from the third cleanup is the purified library (before sequencing adapter is added). Label and save this tube.

STOPPING POINT You can store the eluate from the third cleanup at 4 °C for up to one week or at –20 °C for longer storage.


27

Add the Sequencing Adapter to the Purified Library

Add the Sequencing Adapter to the Purified Library

Retrieve Reagents

Prepare Reagents

Add the sequencing adapter in a post-PCR room.

1 Vortex reagents for 20 sec, and then briefly centrifuge them before use.

2 Combine the components in Table 7 in a new PCR tube to prepare the PCR mix.

Table 7. Reagents for sequencing adapter PCR

3 Store the remaining purified library (before sequencing adapter is added) at –20 °C in case the PCR step needs to be repeated.


TSP Adapter Mix (Fluidigm PN 101-0408)*

* For dual indexing, replace the TSP Adapter Mix with a Dual Index Adapter Mix from the Targeted DNA Seq Library Adapter Set (PN 101-2412). For more information about dual indexing, see Appendix F.

Remove from –20 °C, thaw to room temperature.

PCR Water (Fluidigm PN 101-5941)

DNA Dilution Reagent (Fluidigm PN 100-9167)

4X TSP Master Mix (Fluidigm PN 101-3055)

Agencourt AMPure XP magnetic beads (Beckman Coulter PN A63880)

Remove from storage, keep at room temperature.

DNase-free water

Ethanol, absolute

Component Volume per Reaction (μL)

PCR Water (Fluidigm PN 100-5941) 7.5

4X TSP Master Mix (Fluidigm PN 101-3055) 7.5

TSP Adapter Mix (Fluidigm PN 101-0408)*

* For dual indexing, replace the TSP Adapter Mix with a Dual Index Adapter Mix from the Targeted DNA Seq Library Adapter Set (PN 101-2412). For more information about dual indexing, see Appendix F.

6.0

Purified library 9.0

Total 30.0


28

Clean Up the PCR Product (0.8X SPRI for Solid Tumor Panel, 0.6X SPRI for RNA Fusions Panel)

4 Perform PCR using a stand-alone thermal cycler:

Clean Up the PCR Product (0.8X SPRI for Solid Tumor Panel, 0.6X SPRI for RNA Fusions Panel)Clean up the PCR product (see Add the Sequencing Adapter to the Purified Library on page 28) in a post-PCR room.

IMPORTANT• The quality of PCR products prepared is critical to the success of amplicon sequencing. Any

contamination of primers/tags/adapters or the presence of primer dimers in the PCR products will affect sequencing read quality. Therefore, before sequencing, the sequencing library should be purified and qualified.

• If the 80% ethanol is more than 1 day old, prepare a fresh batch (see Prepare the Reagents for Cleanup on page 24).

1 In a new 1.5 mL microcentrifuge tube, pipet 25 μL of the PCR product into 25 μL of DNase-free water. Mix to dilute the PCR product, and then briefly centrifuge the tube.

2 Suspend magnetic beads in diluted PCR product:

a Ensure that the Agencourt AMPure XP magnetic beads are at room temperature, and then vortex the beads at high speed for 20 sec.

b Pipet the Agencourt AMPure XP magnetic beads into the same tube with the diluted PCR product (see Step 1). Expel any beads left in the pipette tip by pipetting the suspension up and down 10 times.

3 Vortex the suspension at high speed for 20 sec.


Temperature Time Cycles Description

95 ºC 15 min 1 Hot start

95 ºC 15 sec 10 for Solid Tumor Panel

20 for RNA Fusions Panel*

* For the RNA Fusions Panel, the recommended starting point is 20 cycles. The appropriate number of cycles will depend on the number and quality of the samples in the pool and may need to be determined empirically.

PCR

60 ºC 90 sec

68 ºC 90 sec

68 ºC 3 min 1 Final extension

4 °C ∞ 1 Hold

DNA Libraries RNA Libraries

Component Volume forSolid Tumor Panel (μL)

Volume forRNA Fusions Panel (μL)

Diluted PCR product 50 50

Agencourt AMPure XP magnetic beads 40 30

Total 90 80


29

Perform Quality Control on the Sequencing Library (after sequencing adapter is added)









9 Transfer the tubes to a rack and open the tube. Remove any remaining ethanol by drying the beads at 37 °C for 1 min or air-drying the beads at room temperature for 10–15 min.

10 To the dried beads, pipet 45 μL of DNA Dilution Reagent. Vortex the suspension at high speed for 20 sec.



13 Keeping the tube on the magnetic stand, pipet the entire eluate to a new tube. The eluate contains the final sequencing library (after sequencing adapter is added). Label and save this tube.

STOPPING POINT You can store the sequencing library at 4 °C for up to one week or at –20 °C for longer storage.

Perform Quality Control on the Sequencing Library (after sequencing adapter is added)Perform quality control in a post-PCR room.

Quality control includes quantifying an aliquot of the library and performing Agilent Bioanalyzer analysis on aliquots of the purified library (before sequencing adapter is added) and sequencing library (after sequencing adapter is added).

NOTE The expected average size for the Solid Tumor Panel is approximately 300 bp. The expected average size for the RNA Fusions Panel is approximately 300–400 bp.

1 Estimate the concentration of the sequencing library both before and after sequencing adapter is added by quantifying 2 μL of the library with a Qubit Fluorometer following the manufacturer’s instructions. We recommend quantifying the libraries in triplicate.


30

Perform Quality Control on the Sequencing Library (after sequencing adapter is added)

2 Run 1 μL of the sequencing library (after sequencing adapter is added) on an Agilent High Sensitivity DNA Chip following the manufacturer’s instructions. We recommend running the samples in triplicate. When using the Bioanalyzer results to determine library concentration, be sure to include all material between 150 and 1,000 bp in length.

Only trace amounts of primer, if any, should be visible on the electropherogram for the sequencing library (after sequencing adapter is added). Ensure that the library pool passes QC. If the sequencing library (after sequencing adapter is added) does not pass QC, see Appendix H: Troubleshooting on page 44.

Figure 10 shows an example of the expected profile for each panel type.

Figure 10. Sequencing library (undiluted) after sequencing adapter is added.

Solid Tumor Panel

RNA Fusions Panel


31

Sequence the Library

Sequence the LibrarySequence the sequencing library (after sequencing adapter is added) on an Illumina sequencer.

IMPORTANT The sequencer must be able to run 2 x 150-cycle paired-end reads.

After the addition of the sequencing adapter and final cleanup, individual libraries from different panels (Solid Tumor and RNA Fusions) can be combined and sequenced together, if desired. The ratio of each library should be adjusted based on concentration and number of samples to achieve the desired per-sample sequencing depth for each sample and panel. For samples run with the Solid Tumor Panel, a minimum of 1–2 million reads per sample should be targeted. For samples run with the RNA Fusions Panel, a minimum of 1 million total reads per sample should be targeted.


32

Appendix A: LP 8.8.6 IFC Bundled Kit Components

Appendix A: LP 8.8.6 IFC Bundled Kit Components

NOTE Additional LP 8.8.6 IFCs and barrier tape packs can be ordered from fluidigm.com

Appendix B: LP 8.8.6 Reagent Kit ComponentsAdvanta Solid Tumor NGS Assay Pools (PN 101-8816)

Advanta RNA Fusions Reagent Kit (PN 101-8817)

Box Component Part Number Quantity

Advanta Solid Tumor NGS Library Prep Assay—LP 8.8.6, 2 IFCs (PN 101-7033)

Advanta Solid Tumor NGS Assay Pools (8 Pool Set)

101-8816 1 set

Advanta NGS Library Prep Reagent Kit—LP 48.48 & LP 8.8.6, 2 IFCs

101-7663 1 kit

LP 8.8.6 IFC 101-7587 2 IFCs

Juno LP 48.48 & LP8.8.6 Barrier Tape 101-8273 4 tapes

LP Control Line Fluid 48.48 & 8.8.6 (300 μL each)

101-6335 4 syringes

Box Component Part Number Quantity

Advanta RNA Fusions NGS Library Prep Assay—LP 8.8.6, 2 IFCs (PN 101-8654)

Advanta RNA Fusions Reagent Kit 101-8817 1 kit

Advanta NGS Library Prep Reagent Kit—LP 48.48 & LP 8.8.6, 10 IFCs

101-7663 1 kit

LP 8.8.6 IFC 101-7587 2 IFCs

Juno LP 48.48 & LP 8.8.6 Barrier Tape 101-8273 4 tapes

LP Control Line Fluid 48.48 & 8.8.6 (300 μL each)

101-6335 4 syringes

Advanta Solid Tumor NGS Assay Pools:

• 1 tube Pool 1, 30 μL (PN 101-8710)

• 1 tube Pool 2, 30 μL (PN 101-8711)

• 1 tube Pool 3, 30 μL (PN 101-8712)

• 1 tube Pool 4, 30 μL (PN 101-8713)

• 1 tube Pool 5, 30 μL (PN 101-8714)

• 1 tube Pool 6, 30 μL (PN 101-8715)

• 1 tube Pool 7, 30 μL (PN 101-8716)

• 1 tube Pool 8, 30 μL (PN 101-8717)

Advanta RNA Fusions NGS Assay Pools:

• 1 tube Pool 1, 30 μL (PN 101-8646)

• 1 tube Pool 2, 30 μL (PN 101-8647)

• 1 tube Pool 3, 30 μL (PN 101-8648)

• 1 tube Pool 4, 30 μL (PN 101-8649)

• 1 tube Pool 5, 30 μL (PN 101-8650)

• 1 tube Pool 6, 30 μL (PN 101-8651)

• 1 tube Pool 7, 30 μL (PN 101-8652)

• 1 tube Pool 8, 30 μL (PN 101-8653)

1 tube 5X TSP RT Buffer, 120 μL (PN 101-8913)

1 tube TSP Reverse Transcriptase, 30 μL (PN 101-8818)

1 tube TSP RNase Inhibitor, 12 μL (PN 101-8911)


33

http://www.fluidigm.com

Appendix B: LP 8.8.6 Reagent Kit Components

Advanta NGS Library Prep Reagent Kit—LP 48.48 & 8.8.6, 2 IFCs (PN 101-7663)

1 tube TSP Sample Loading Reagent v2, 200 μL (PN 101-7634)

1 tube TSP Adapter Mix, 120 μL (PN 101-0408)

1 tube PCR Water, 1.8 mL (PN 100-5941)

1 bottle DNA Dilution Reagent, 3.0 mL (PN 100-9167)

1 bottle TSP Harvest Reagent, 10 mL (PN 101-0743)

1 tube TSP Assay Loading Reagent, 75 μL (PN 101-0409)

1 tube 4X TSP Master Mix, 210 μL (PN 101-3055)

1 tube TSP DNA Polymerase, 50 μL (PN 101-0995)


34

Appendix C: Map of the LP 8.8.6 IFC

Appendix C: Map of the LP 8.8.6 IFC

How to Use the Map

1 Print this page on a color printer at actual size (100% scale).

2 Place the printed map under the LP 8.8.6 IFC and align the notched A1 corner at the upper left of the IFC with the pink corner of the map.

3 If necessary, use clear tape to anchor the IFC to the printed map.

4 Follow the procedures to pipet reagents into the IFC (page 19) or harvest samples from the IFC (page 22).

Samplemixes

Assaymixes andnegative assay mix

TSP HarvestReagent

TSP HarvestReagent

ControlLine Fluid

ControlLine Fluid

LP 8.8.6 IFC


35

Appendix D: LP 8.8.6 IFC Scripts

Appendix D: LP 8.8.6 IFC ScriptsJuno scripts for the LP 8.8.6 IFC (156x):

Appendix E: LP 8.8.6 IFC One Step Thermal ProtocolThe One Step LP—8.8.6 script contains these thermal cycling protocols:

Type of Script Script Description

Basic Prime LP—8.8.6 Preparation of the LP 8.8.6 IFC for loading

One Step LP—8.8.6 One-step load mix, PCR, and harvest ofLP 8.8.6 IFC

Temperature Time Cycles Description

65 ºC 18 min 1 Thermal mix

95 ºC 15 min 1 Hot start

95 ºC 15 sec 17 PCR

60 ºC 90 sec

68 ºC 90 sec

68 ºC 3 min 1 Final extension

10 ºC Until harvest 1 Hold


36

Appendix F: Dual Indexing

Appendix F: Dual IndexingUse dual indexing if you are processing more than 384 samples. Dual indexing using theTargeted DNA Seq Library Adapter Set (PN 101-2412) adds a second (i5) barcode next to theP5 sequencing adapter, allowing you to generate 385–1,536 unique barcoded libraries per run.

The sequencing workflow for reading of a second index varies among Illumina sequencers. When preparing a sample sheet, enter the appropriate i5 sequence for your sequencer according to Table 8:

Table 8. Targeted DNA Seq Library Adapter Set i5 sequences

IMPORTANT When you create your sample sheet in the Illumina Experiment Manager, select Nextera® for adapter trimming purposes.

Kit Components of the Targeted DNA Seq Library Adapter Set (PN 101-2412)

IFC Type Numberof IFCs

Number ofSamples

Number ofUnique On-IFC

(i7) Barcodes

Number ofUnique Forward

(i5) Barcodes

EstimatedAverage Read

Depth per Sample

Estimated AverageRead Depth per

Amplicon per Sample

LP 8.8.6 16 768 384 2 19,500 260

16 768 192 4 19,500 260

32 1,536 384 4 9,700 130

P7

BC (i7)

Tag 2

P5 Tag 2

FBC (i5)

P7

BC (i7)

Tag 2TSP Adapter Mix Dual Index Adapter Mix

P5 Tag 2Target sequenceTarget sequence

4 x 384 barcodes

Dual indexing384 barcodes

Single indexing

Dual Index Adapter Mix (i5 Index)

i5 Sequence for Sample Sheet (MiSeq™, HiSeq® 2000/2500)

i5 Sequence for Sample Sheet(MiniSeq™, NextSeq™, HiSeq 3000/4000)

1 (PN 101-2037) TATGAGTGAT ATCACTCATA

2 (PN 101-2038) ATATGACTGT ACAGTCATAT

3 (PN 101-2039) GACTATACAT ATGTATAGTC

4 (PN 101-2040) GATACTATGT ACATAGTATC

1 tube Dual Index Adapter Mix 1, 120 μL (PN 101-2037)





37

Appendix G: TSP Library Barcode Plates

Appendix G: TSP Library Barcode PlatesThe Targeted DNA Seq Barcode Plates (PN 101-0744) are supplied in four 96-well plates. Use a barcode as a unique identifier of a genomic sample.

Plate Maps

TSP Barcode Plate 1 (PN 101-0736)


1 2 3 4 5 6 7 8 9 10 11 12

A TSP0001 TSP0009 TSP0017 TSP0025 TSP0033 TSP0041 TSP0049 TSP0057 TSP0065 TSP0073 TSP0081 TSP0089

B TSP0002 TSP0010 TSP0018 TSP0026 TSP0034 TSP0042 TSP0050 TSP0058 TSP0066 TSP0074 TSP0082 TSP0090

C TSP0003 TSP0011 TSP0019 TSP0027 TSP0035 TSP0043 TSP0051 TSP0059 TSP0067 TSP0075 TSP0083 TSP0091

D TSP0004 TSP0012 TSP0020 TSP0028 TSP0036 TSP0044 TSP0052 TSP0060 TSP0068 TSP0076 TSP0084 TSP0092

E TSP0005 TSP0013 TSP0021 TSP0029 TSP0037 TSP0045 TSP0053 TSP0061 TSP0069 TSP0077 TSP0085 TSP0093

F TSP0006 TSP0014 TSP0022 TSP0030 TSP0038 TSP0046 TSP0054 TSP0062 TSP0070 TSP0078 TSP0086 TSP0094

G TSP0007 TSP0015 TSP0023 TSP0031 TSP0039 TSP0047 TSP0055 TSP0063 TSP0071 TSP0079 TSP0087 TSP0095

H TSP0008 TSP0016 TSP0024 TSP0032 TSP0040 TSP0048 TSP0056 TSP0064 TSP0072 TSP0080 TSP0088 TSP0096

1 2 3 4 5 6 7 8 9 10 11 12










38




1 2 3 4 5 6 7 8 9 10 11 12









1 2 3 4 5 6 7 8 9 10 11 12










39


Barcode Lists

TSP Barcodes Plate 1

Name Sequence Well Name Sequence Well Name Sequence Well

TSP0001 GTCGTCGTCT A1 TSP0033 TCGAGGTACT A5 TSP0065 TCTCAGTTCT A9

TSP0002 GATGTAGCGT B1 TSP0034 CTAAGTCATG B5 TSP0066 GAGTTATCAG B9

TSP0003 GTGCTGTCGT C1 TSP0035 CTGTTCTAGC C5 TSP0067 GTAACTAGCA C9

TSP0004 TGAGCGTGCT D1 TSP0036 GATCCTGAGC D5 TSP0068 GTGTGACTAA D9

TSP0005 TGTGTGCATG E1 TSP0037 GTCGGTCTGA E5 TSP0069 GCAGTTGATT E9

TSP0006 GAGTGCATCT F1 TSP0038 CAGATGTCCT F5 TSP0070 TCACTAAGCT F9

TSP0007 CAGTCAGAGT G1 TSP0039 CACTGCTTGA G5 TSP0071 CATGCGGAGA G9

TSP0008 GTATGAGCAC H1 TSP0040 CTTACGTTGC H5 TSP0072 CACTCGATAA H9

TSP0009 TCTCTGTGCA A2 TSP0041 TCCTTGTTCT A6 TSP0073 GAGTAGCCTG A10

TSP0010 GCACGTAGCT B2 TSP0042 GAAGTCAAGC B6 TSP0074 CTGATGACTT B10

TSP0011 TGTCTCTATC C2 TSP0043 GCGCATTATG C6 TSP0075 TCGAGTCGGA C10

TSP0012 TATCTCATGC D2 TSP0044 GACAGCAAGC D6 TSP0076 TGATACCACT D10

TSP0013 TATCGATGCT E2 TSP0045 TCACGACGAA E6 TSP0077 TGACTCAATG E10

TSP0014 TGATACTCTG F2 TSP0046 TATGTGCCGT F6 TSP0078 GGTATGGACG F10

TSP0015 TCAGCGATAT G2 TSP0047 GCCGTGATGT G6 TSP0079 TTGTGACGCA G10

TSP0016 GTAGTACACA H2 TSP0048 GGTGTGTAAG H6 TSP0080 GCGCTCTATT H10

TSP0017 CATGATACGC A3 TSP0049 GTATTGCTGC A7 TSP0081 TATGACTCGA A11

TSP0018 TGATGTATGT B3 TSP0050 CTGAGCTTCT B7 TSP0082 TGTCATCAAG B11

TSP0019 TTGTTGCTGT C3 TSP0051 TGCCAGTGTC C7 TSP0083 CATGCGCTTA C11

TSP0020 TGTTGTGGTA D3 TSP0052 CTGAATGCTG D7 TSP0084 TTCGACTTGA D11

TSP0021 GTTGATGAGT E3 TSP0053 TGATAGCCTC E7 TSP0085 TCTCTGCGAT E11

TSP0022 CGTCTTCTTA F3 TSP0054 CGTTGAGTGC F7 TSP0086 GCTTACATTG F11

TSP0023 CTCTTAGTTC G3 TSP0055 GCTATGGTCA G7 TSP0087 GCTTCACTAG G11

TSP0024 TGGTGTCCGT H3 TSP0056 CTGGTGGACT H7 TSP0088 TCAAGAGTGC H11

TSP0025 TTCTCATCGT A4 TSP0057 CTCGACGTTA A8 TSP0089 GATAGGCACA A12

TSP0026 TTGTCCTTGC B4 TSP0058 TCTTCGGTGG B8 TSP0090 TACGATGCCA B12

TSP0027 CGTAATGAGC C4 TSP0059 GCGGTATCTC C8 TSP0091 GTACAGCAAT C12

TSP0028 GTGGCTTCGT D4 TSP0060 CGCATTATCA D8 TSP0092 CTAGGCTACA D12

TSP0029 CGAATGTATG E4 TSP0061 TCCTAGATGA E8 TSP0093 GGTAGGCTAC E12

TSP0030 TGTCAGCTTA F4 TSP0062 TTCTCTTAGC F8 TSP0094 TCCGACAGTG F12

TSP0031 TTACACGTTC G4 TSP0063 TTGGTAGATG G8 TSP0095 GAACACTCTG G12

TSP0032 TGACTAGCTT H4 TSP0064 CTTGTGCATA H8 TSP0096 TCACACTTAG H12


40




TSP0097 GTATCGTCGT A1 TSP0129 GCGTCTGAAT A5 TSP0161 CGAGGAGAAC A9

TSP0098 GTCGTGTACT B1 TSP0130 TTGCTTAGTC B5 TSP0162 CATCTGCTAA B9

TSP0099 TCAGTGTCTC C1 TSP0131 TCTTGTTCAC C5 TSP0163 CTGTAGATTC C9

TSP0100 CTGTGTCGTC D1 TSP0132 GAAGGAGATA D5 TSP0164 GGATCTCATA D9

TSP0101 GATGCGAGCT E1 TSP0133 GTTCTTCGTA E5 TSP0165 TGCTCATTGA E9

TSP0102 CGCAGTCTAT F1 TSP0134 TCGAATGTGC F5 TSP0166 TCGCATAAGT F9

TSP0103 CGAGTGCTGT G1 TSP0135 TGCGGATGGT G5 TSP0167 TGCTTCAGAT G9

TSP0104 TATAGCACGC H1 TSP0136 TGTTACGATC H5 TSP0168 TGCAATGTGT H9

TSP0105 TATGCGCTGC A2 TSP0137 GTCTTGGCTC A6 TSP0169 GATTGTTCTC A10

TSP0106 CGCGTATCAT B2 TSP0138 TTACGCAGTG B6 TSP0170 GATGACTGTT B10

TSP0107 TACTGAGCTG C2 TSP0139 GTATAACGCT C6 TSP0171 GATGAATAGC C10

TSP0108 GACGTCTGCT D2 TSP0140 GAAGCGCACT D6 TSP0172 TCATTGCTAG D10

TSP0109 CGTATGATGT E2 TSP0141 GGAGACTGTA E6 TSP0173 TCTTCGTAGA E10

TSP0110 CTAGATCTGA F2 TSP0142 GCTCTAACAT F6 TSP0174 CATGTTGTTG F10

TSP0111 TATCAGTCTG G2 TSP0143 TTGTCGAGAC G6 TSP0175 CGATACGTGT G10

TSP0112 TCAGATGCTA H2 TSP0144 TATCCGTGTC H6 TSP0176 GCTTATCGAT H10

TSP0113 TCATATCGCG A3 TSP0145 CGTGTCCAGT A7 TSP0177 TATAGAGCCG A11

TSP0114 GCACGCGTAT B3 TSP0146 TGTGCCATCT B7 TSP0178 CTTAGAGTCG B11

TSP0115 TGATAGAGAG C3 TSP0147 CTGCAAGTCT C7 TSP0179 GATGCAGCCA C11

TSP0116 CTCAGCAGTG D3 TSP0148 GTGTCGAAGA D7 TSP0180 GCTCTCTAGG D11

TSP0117 TACTGCAGCG E3 TSP0149 CTGTCGGATG E7 TSP0181 GAGTATCGCC E11

TSP0118 CACATACAGT F3 TSP0150 CTTGAGCGTC F7 TSP0182 TCTAGGAGTG F11

TSP0119 GAGACTATGC G3 TSP0151 CACGGAGTAT G7 TSP0183 TATGGATGAG G11

TSP0120 CGACGCTGAT H3 TSP0152 TGTAGGACAC H7 TSP0184 TGAGCACGTC H11

TSP0121 GACGATCGCA A4 TSP0153 CCGTTCGTTG A8 TSP0185 CGGTAGACAT A12

TSP0122 TCTACGACAT B4 TSP0154 GGTCACATCA B8 TSP0186 CGACAATCTA B12

TSP0123 CTACATACTA C4 TSP0155 CTGGAGCGAT C8 TSP0187 TGATTGCGAC C12

TSP0124 CAGCATCTAG D4 TSP0156 CCGTCTATAG D8 TSP0188 GACGCAGCTT D12

TSP0125 TAGGTGGAAT E4 TSP0157 GTTAGCTTCG E8 TSP0189 CATGGCACTA E12

TSP0126 CTCGTTATTC F4 TSP0158 GTCACGTGGT F8 TSP0190 CAGGCAGATC F12

TSP0127 GGTGTTAGTG G4 TSP0159 TATCCGCATG G8 TSP0191 CGCGATACTT G12

TSP0128 GTTCGATTGT H4 TSP0160 GACTACTCGT H8 TSP0192 GAGGACGAAG H12


41




TSP0193 GTGTATGCGT A1 TSP0225 GTACTAAGAG A5 TSP0257 TGATGCATTC A9

TSP0194 GAGTGATCGT B1 TSP0226 GTCCAGACAT B5 TSP0258 TGAGATTGGA B9

TSP0195 CGCTGTAGTC C1 TSP0227 GAGACCTCTA C5 TSP0259 GCTTGCATAT C9

TSP0196 CATGTCGTCA D1 TSP0228 GACAGGTGAC D5 TSP0260 GACTCTTCAT D9

TSP0197 GAGTGTCACT E1 TSP0229 CATACCTGAT E5 TSP0261 GTTGACATTC E9

TSP0198 TGCGTAGTCG F1 TSP0230 GTTGTCGCTT F5 TSP0262 TTAGAGATGG F9

TSP0199 CTGTACGTGA G1 TSP0231 GTGGTTAGGT G5 TSP0263 GCTCAATAAC G9

TSP0200 CTGCATGATC H1 TSP0232 CCGTATGCTA H5 TSP0264 TAGCATGGAT H9

TSP0201 TAGTAGCGCG A2 TSP0233 TTGTTGGCGG A6 TSP0265 TTCAGTCGCG A10

TSP0202 TAGTCTGTCA B2 TSP0234 CGTGTCTTAG B6 TSP0266 CCTGATGTGC B10

TSP0203 CATGCATCAT C2 TSP0235 CAGCGTGTCT C6 TSP0267 TAGCCTATAC C10

TSP0204 CTAGCAGATG D2 TSP0236 GACTTCGCTA D6 TSP0268 TCAGAGTCAT D10

TSP0205 CACGAGATGA E2 TSP0237 GGACTAGGTA E6 TSP0269 TCGTTCAGAC E10

TSP0206 TCGATATCTA F2 TSP0238 TGCGATGAGG F6 TSP0270 CCATGTGTCG F10

TSP0207 TACATGATAG G2 TSP0239 GATTCAGCTC G6 TSP0271 TGAACTACGA G10

TSP0208 TGTAGGTGGA H2 TSP0240 GTCGAGGATC H6 TSP0272 TCGCAACAAT H10

TSP0209 TTAGTGGTGA A3 TSP0241 GTCGAAGCTG A7 TSP0273 GTAGCATTCT A11

TSP0210 TGTGAATCTC B3 TSP0242 GGACGTGACT B7 TSP0274 CAGCCGATGT B11

TSP0211 CGTTAGCGTA C3 TSP0243 CGCTAGGAGA C7 TSP0275 GTCGTTAGAG C11

TSP0212 TACTAGGATC D3 TSP0244 TCACTGGAGG D7 TSP0276 GCATCTATTG D11

TSP0213 GATGAGGTAT E3 TSP0245 GGAGATCGAT E7 TSP0277 GCATTCGTAC E11

TSP0214 TATGGTAAGG F3 TSP0246 CTCTAGGTAT F7 TSP0278 CGGACTCTAG F11

TSP0215 CTTAGTTCGC G3 TSP0247 CTGTACTGTT G7 TSP0279 CTATGACACT G11

TSP0216 TCATTCAGTG H3 TSP0248 CTAGCTCATT H7 TSP0280 GATGCAATCG H11

TSP0217 CCATATGATC A4 TSP0249 GGATATACAC A8 TSP0281 GTGCACGAAT A12

TSP0218 TCGCTGAACA B4 TSP0250 CGTTGTTCTA B8 TSP0282 CGAAGTGCAC B12

TSP0219 GAACTATCAC C4 TSP0251 TAGCGTATTG C8 TSP0283 TCTGACCAGT C12

TSP0220 CTGCGAATGT D4 TSP0252 TAGCACATTC D8 TSP0284 TAGACTCCAT D12

TSP0221 TGGATGACAT E4 TSP0253 TCTAGTATCC E8 TSP0285 GCGAACGTAC E12

TSP0222 TTCGTTCCTG F4 TSP0254 CGATCTGTAT F8 TSP0286 TGCCTTCTTG F12

TSP0223 GGACAGATGG G4 TSP0255 CACTATGGAT G8 TSP0287 TCATACGGTA G12

TSP0224 GGAACACAGG H4 TSP0256 TATGCACTCT H8 TSP0288 TAGATCTACC H12


42




TSP0289 GCGTCGTGTA A1 TSP0321 TGGAGCATGT A5 TSP0353 TAGATCGAAG A9

TSP0290 CGCTATCAGT B1 TSP0322 TTGTCACATC B5 TSP0354 CATGAGTACT B9

TSP0291 GTGCTCATGT C1 TSP0323 TTCGAGCTAT C5 TSP0355 GTAAGTACTG C9

TSP0292 GTCTACTGTC D1 TSP0324 GGTCGTGCAT D5 TSP0356 TGGACAGTAT D9

TSP0293 GTATCTCTCG E1 TSP0325 CGAGCATTGT E5 TSP0357 GTCTTAGACT E9

TSP0294 TACATCGCTG F1 TSP0326 TTGCACTCAG F5 TSP0358 CTGATATCCG F9

TSP0295 GACTGTACGT G1 TSP0327 CTGACAAGTG G5 TSP0359 TCTCGTACAA G9

TSP0296 TAGAGTCTGT H1 TSP0328 CACGAAGAGC H5 TSP0360 GCATTCACGT H9

TSP0297 CGTCTATGAT A2 TSP0329 GACGTGCTTC A6 TSP0361 GAGTCCGACT A10

TSP0298 CAGCTATAGC B2 TSP0330 TGGAGGTGTT B6 TSP0362 CGACGTTATC B10

TSP0299 TGCGAGACGT C2 TSP0331 TGGAGTCATC C6 TSP0363 TAGGTCTATG C10

TSP0300 GCGTAGACGA D2 TSP0332 GTAATGTGCG D6 TSP0364 TCGTAGACCT D10

TSP0301 CGCAGAGCAT E2 TSP0333 TGTGAGGTAC E6 TSP0365 GTATTCAGCA E10

TSP0302 TGTACAGCGA F2 TSP0334 TGTCAAGAGC F6 TSP0366 CAGTGGACGT F10

TSP0303 GCAGCTGTCA G2 TSP0335 CTATAAGCGC G6 TSP0367 TCTTCTATCG G10

TSP0304 TCATCATGCG H2 TSP0336 CGAAGCGTGA H6 TSP0368 CGGATAGCTG H10

TSP0305 GATATATGTC A3 TSP0337 GAGTCGATCC A7 TSP0369 TCTCATGTTG A11

TSP0306 GTGAAGGTAA B3 TSP0338 GAAGTCGCTG B7 TSP0370 TGGCACTATG B11

TSP0307 CTAATCGTGT C3 TSP0339 CTAAGCACGT C7 TSP0371 TATGGCACAT C11

TSP0308 GGTGTCTTGT D3 TSP0340 GTAGATGGAC D7 TSP0372 TGACTTCTGA D11

TSP0309 CCTCGTTGTT E3 TSP0341 GCTGTCCGTA E7 TSP0373 CAGAGTTGTT E11

TSP0310 GTCTCAATGT F3 TSP0342 TGAGCCACAT F7 TSP0374 TGCAAGCTCA F11

TSP0311 CATTCTCTGA G3 TSP0343 CTGTATACCA G7 TSP0375 GCTAGCTACT G11

TSP0312 GGCTGTGATC H3 TSP0344 CACTTCAGTA H7 TSP0376 GTCTTGCTTG H11

TSP0313 CTCAATCGTA A4 TSP0345 TCCATCGTAT A8 TSP0377 CGATCGAGGT A12

TSP0314 CGCTAATGTA B4 TSP0346 GTTGTCTTAC B8 TSP0378 TCAGTACAGG B12

TSP0315 TTCTGTTGCC C4 TSP0347 CATTGCAGTT C8 TSP0379 TGAGGCGACT C12

TSP0316 CCTGTGTAGA D4 TSP0348 GATTGATTGC D8 TSP0380 TATCCGACGT D12

TSP0317 GATAAGAAGG E4 TSP0349 CTAGTAGCTT E8 TSP0381 GCGAATACTG E12

TSP0318 CAGGTCACAT F4 TSP0350 GTATAGATCC F8 TSP0382 GATCTGCAAC F12

TSP0319 TCTGCCTATA G4 TSP0351 CATCATAGTC G8 TSP0383 TTCGAGCAGC G12

TSP0320 TGTTCGATAG H4 TSP0352 TAAGTACGCA H8 TSP0384 CTACGGAGCT H12


43

Appendix H: Troubleshooting

Appendix H: TroubleshootingFor troubleshooting, preventive maintenance, and instructions for disposal of consumables, see the Juno System User Guide (100-7070).

Observations Possible Causes Recommended Actions

Low yield: The purified library (before sequencing adapter is added) is <0.2 ng/μL as measured by Qubit.

• Low sample quality

• Low input concentration of DNA

• Loss of sample during cleanup

• Confirm that genomic DNA input is at least 12.5 ng or that RNA input is at least 10 ng.

• Check sample quality. For FFPE or other degraded samples, input can be increased to up to 100 ng. Increasing input of low quality samples in this range will improve sample performance.

• Confirm that sample mixes and 10X assay pools for LP 8.8.6 IFC have been prepared as described in Prepare the Assay Mixes and Sample Mixes on page 13.

• Repeat SPRI cleanup following the recommended protocol: Prepare fresh 80% ethanol, pipet slowly, ensure appropriate pipetting volumes, mix well. Do not disturb or aspirate the beads. Cleanup may require practice for reproducible performance.

Low yield: The sequencing library (after sequencing adapter is added) is <1 ng/μL as measured by Qubit.

• Insufficient number of PCR cycles used during adapter PCR.

• Failure of sequencing adapter addition PCR reaction

• Loss of sample during cleanup

• Repeat adapter PCR reaction with the purified harvest material.

• Confirm that the PCR reaction mix was prepared as described in Add the Sequencing Adapter to the Purified Library on page 28.

• Confirm that the appropriate thermal conditions were used.

• Repeat SPRI cleanup following the recommended protocol: Prepare fresh 80% ethanol, pipet slowly, ensure appropriate pipetting volumes, mix well. Do not disturb or aspirate the beads. Cleanup may require practice for reproducible performance.

Primer present in sequencing library: The sequencing library (after P5 sequencing adapter is added) shows a small spike immediately after the lower marker.

• Mispipetting of the sequencing adapter.

• Incomplete cleanup of the library after sequencing adapter addition.

Repeat the cleanup of the PCR product after sequencing adapter addition and repeat the Bioanalyzer analysis.

IMPORTANT Before repeating the cleanup, add 5 μL of DNA Dilution Reagent to the PCR product to bring the volume up to 50 μL.


44

Appendix I: Related Documents

Appendix I: Related DocumentsGo to fluidigm.com to download these related documents.

Appendix J: SafetyGeneral Safety

In addition to your site-specific safety requirements, Fluidigm recommends the following general safety guidelines in all laboratory and manufacturing areas:

• Use the appropriate personal protective equipment (PPE): safety glasses, fully enclosed shoes, lab coats, and gloves, according to your laboratory safety practices.

• Know the locations of all safety equipment (fire extinguishers, spill kits, eyewashes/showers, first-aid kits, safety data sheets, etc.), emergency exit locations, and emergency/injury reporting procedures.

• Do not eat, drink, or smoke in lab areas.

• Maintain clean work areas.

• Wash hands before leaving the lab.

Sample dropout by sequencing: A sample has very low reads compared with other samples.

• Sample loading failure due to bubbles

• Insufficient volume in one or more sample inlets

• Dropout sample was of low quality

• Ensure that the assay and sample mixes were centrifuged at 3000 x g for 5 min prior to reagent transfer.

• Ensure that there are no bubbles in the sample plates before transferring sample mixes into the IFC. Remove bubbles as necessary.

• Ensure that appropriate pipetting volumes are transferred into the IFC inlets.

• Do not go beyond the first stop of the pipette when transferring reagents into the IFC inlets.

• Determine sample quality using standard procedures for RNA or DNA. For low quality samples, input can be increased up to 100 ng. A PCR based method that measures amplifiable nucleic acid content in the 100–200 bp range is recommended

Sequencing results: Higher than expected reads for customer provided positive control fusion product(s) in other samples.

Carryover of high abundance fusion products during library cleanup and adapter addition.

Reduce the input amount of positive control samples with high expression of fusion products. Reads assigned to a positive control fusion event should be less than 10% of the total number of sequencing reads.

Observations Possible Causes Recommended Actions

Title Part Number

Library Preparation with the LP 8.8.6 IFC Quick Reference 101-8811

Control Line Fluid Loading Procedure 68000132

Juno System User Guide 100-7070


45

http://www.fluidigm.com

Instrument Safety

For complete instrument safety information, including a full list of the symbols on the instrument, refer to the Juno System User Guide (100-7070).

WARNING BIOHAZARD. If you are putting biohazardous material on the instrument, use appropriate personal protective equipment and adhere to Biosafety in Microbiological and Biomedical Laboratories (BMBL), a publication from the Centers for Disease Control and Prevention, and to your lab's safety protocol to limit biohazard risks. If biohazardous materials are used, properly label the equipment as a biohazard. For more information, see the BMBL guidelines online at cdc.gov/biosafety/publications/index.htm.

Chemical Safety

The responsible individuals must take the necessary precautions to ensure that the surrounding workplace is safe and that instrument operators are not exposed to hazardous levels of toxic substances. When working with any chemicals, refer to the applicable safety data sheets (SDSs) provided by the manufacturer or supplier.

Disposal of Products

Used IFCs and reagents should be handled and disposed of in accordance with federal, state, regional, and local laws for hazardous waste management and disposal.

For technical support visit techsupport.fluidigm.com. For general support visit fluidigm.com/support.North America +1 650 266 6100 | Toll-free (US/CAN): 866 358 4354 | [email protected] Latin America +1 650 266 6100 | [email protected]

Europe/Middle East/Africa/Russia +44 1223 598100 | [email protected] China (excluding Hong Kong) +86 21 3255 8368 | [email protected]

Japan +81 3 3662 2150 | [email protected] All other Asian countries/India/Australia +1 650 266 6100 | [email protected]

For Research Use Only. Not for use in diagnostic procedures. Information in this publication is subject to change without notice. Patent and license information: fluidigm.com/legalnotices. Trademarks: Fluidigm, the Fluidigm logo, Advanta, and Juno are trademarks and/or registered trademarks of Fluidigm Corporation in the United States and/or other countries. All other trademarks are the sole property of their respective owners. © 2020 Fluidigm Corporation. All rights reserved. 02/2020

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