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Ion Sphere™ Particles Quality Assessmentfor the Ion Proton™ and Ion S5™ SystemsUsing the Guava™ easyCyte 5 Benchtop Flow CytometerPublication Number MAN0007496 Revision B.0

■ Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1■ Required materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2■ Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2■ Set up the Guava™ easyCyte 5 Benchtop Flow Cytometer . . . . . . . . . . . . . . . . . . . 3■ Prepare the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4■ Measure the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5■ Analyze the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8■ Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11■ Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

WARNING! Read the Safety Data Sheets (SDSs) and follow the handlinginstructions. Wear appropriate protective eyewear, clothing, and gloves. SafetyData Sheets (SDSs) are available from thermofisher.com/support.

OverviewThis user bulletin describes how to use the Guava™ easyCyte 5 Benchtop FlowCytometer for quality assessment of unenriched and enriched template-positive IonSphere™ Particles (ISPs) prepared with the Ion OneTouch™ 2 Instrument or theIon Chef™ Instrument for the following systems:

• Ion Proton™ System• Ion S5™ System• Ion S5™ XL System

USER BULLETIN

For Research Use Only. Not for use in diagnostic procedures.

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Required materials and equipmentUnless otherwise indicated, all materials are available through http://www.thermofisher.com, MLS: Fisher Scientific (fisherscientific.com) or other majorlaboratory supplier.

Item Source

Guava™ easyCyte 5 Benchtop Flow Cytometer Base System EMD Millipore0500-5005

Microtube (Guava™ tube), 1.5 mL Fisher Scientific02-681-339 or MLS

Screw cap Fisher Scientific02-681-358 or MLS

PBS, 1X MLS

Tween™ 20, molecular grade MLS

SYBR™ Green I Nucleic Acid Gel Stain, 10,000X Concentrate inDMSO

S7563

Nuclease-free Water AM9938 or MLS

Before you begin1. Dilute the SYBR™ Green I Nucleic Acid Gel Stain (10,000X concentrate in DMSO)

to 100X with Nuclease-free Water.

2. Prepare Guava™ Buffer: 1X PBS, 0.05% Tween™ 20.

Ion Sphere Particles (ISPs) Quality Assessment Using Guava™ easyCyte™ 5 Flow CytometerRequired materials and equipment

2 Ion Sphere™ Particles Quality Assessment User Bulletin

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Set up the Guava™ easyCyte 5 Benchtop Flow Cytometer1. Select Guava™ Express Pro Software from the main menu.

2. Enter the set of parameter values that are listed in the following table.

Parameter Setting

FSC Gain ×256

SSC ×8

GRN ×2

Threshold Channel GRN

Threshold Level 5 units

Use High Calibration Voltage Yes for all channels

Flow rate Low (0.24 µL/s)

3. In the left panel, select the following settings:

Parameter Setting

Threshold Parameter GRN

Area Width Parameter NONE

Count Gate All Events

Refresh Rate Cumulative

4. Save the settings for use in this protocol.

Ion Sphere Particles (ISPs) Quality Assessment Using Guava™ easyCyte™ 5 Flow CytometerSet up the Guava™ easyCyte 5 Benchtop Flow Cytometer

Ion Sphere™ Particles Quality Assessment User Bulletin 3

Prepare the samplesNote: Eppendorf LoBind™ microcentrifuge tubes or standard 1.5-mL microcentrifugetubes can be used to prepare and dilute the ISP samples. Use screw-cap 1.5-mLGuava™ tubes for running fully diluted samples on the Guava™ easyCyte 5 BenchtopFlow Cytometer.

Use the following procedure if you used the Ion OneTouch™ 2 System to prepare ISPsfor sequencing on the Ion Proton™ System, or the Ion S5™/Ion S5™ XL System.

1. If you have not done so already, transfer 2 µL of the ISP suspension to a 1.5-mLtube:

Sample type Sample volume and source

Unenriched(After recoveringand washing theISPs)

Transfer a 2‑µL aliquot from the 1 mL of unenriched ISPs inNuclease-free Water to the 1.5‑mL Guava™ tube [see "Washthe template‑positive Ion PI™ Ion Sphere™ Particles" in thefollowing documents:

• Ion PI™ Hi‑Q™ OT2 200 Kit User Guide (Pub. No.MAN0010857)

• Ion 520™ & Ion 530™ Kit – OT2 User Guide (Pub. No.MAN0010844)

• Ion 540™ Kit – OT2 User Guide (Pub. No. MAN0010850)

Unenriched(After transferringthe ISPs to Well 1 ofthe 8‑well strip)

Prepare according to step 4 of "Fill the 8‑well strip" in thefollowing documents:

• Ion PI™ Hi‑Q™ OT2 200 Kit User Guide (Pub. No.MAN0010857)

• Ion 520™ & Ion 530™ Kit – OT2 User Guide (Pub. No.MAN0010844)

• Ion 540™ Kit – OT2 User Guide (Pub. No. MAN0010850)

Transfer a 2‑µL aliquot from the 100 µL of unenriched ISPs inIon OneTouch™ Wash Solution to the 1.5‑mL tube.

Enriched Transfer a 2‑µL aliquot from the 100 µL of enriched ISPs inNuclease‑free Water to the 1.5‑mL tube.

2. Add 2 µL of 100X SYBR™ Green I Nucleic Acid Gel Stain to the sample, pipet upand down to mix, then incubate for ~2 minutes.

3. Add 196 µL of Guava™ Buffer (1X PBS, 0.05% Tween™ 20) to the sample to obtaina total volume of 200 µL. Vortex to mix.

4. In a new 1.5-mL Guava™ tube, further dilute the sample by transferring 2 µL ofstained beads from step 3 into 198 µL of Guava™ Buffer to obtain a total volumeis 200 µL.

Note: The sample dilution factor is 1:10,000.

For ISPs preparedwith theIon OneTouch™ 2System

Ion Sphere Particles (ISPs) Quality Assessment Using Guava™ easyCyte™ 5 Flow CytometerPrepare the samples


Use the following procedure if you used the Ion Chef™ System to prepare ISPs forsequencing on the Ion Proton™ System, or the Ion S5™/Ion S5™ XL System.

1. Transfer an aliquot of ISP suspension to a 1.5-mL tube:

Sample type [1] Positions[2] Sample volume and source

Unenriched A and B of theChef Reagentscartridge[3]

Transfer 5-µL aliquots of ISPs from Reagentscartridge positions A and B to two 1.5-mLtubes.

Enriched A and E of theEnrichmentCartridge v2

Transfer 5-µL aliquots of ISPs from EnrichmentCartridge positions A and E to two 1.5-mLtubes.

[1] See an Ion Chef™ reagent user guide for detailed instructions on collecting quality control samples.[2] Unenriched ISPs in Position A of the Reagents cartridge, and enriched ISPs in Position E of Enrichment

Cartridge v2 correspond with the sample library loaded in Position A of the Reagents cartridge. Unenriched ISPs in Position B of the Reagents cartridge, and enriched ISPs in Position A of Enrichment Cartridge v2 correspond with the sample library loaded in Position B of the Reagents cartridge.

[3] Ion PI™ Hi‑Q™, Ion 510™ & Ion 520™ & Ion 530™, Ion 520™ & Ion 530™, or Ion 540™ Chef Reagents cartridge

2. Add 2 µL of 100X SYBR™ Green I Nucleic Acid Gel Stain and 93 µL Guava™

Buffer to the samples, pipet up and down to mix, then incubate for 2 minutes.

3. Dilute the samples as follows:

Sample type Dilution method

Unenriched In a new 1.5‑mL Guava™ tube, dilute 2 µL of the sample from step 2with 198 µL Guava™ Buffer to obtain a total volume of 200 µL.Vortex to mix.

Enriched In a new 1.5‑mL Guava™ tube, dilute 10 µL of the sample from step2 with 190 µL Guava™ Buffer to obtain a total volume of 200 µL.Vortex to mix.

Note: The Ion Chef™ Instrument performs an on deck dilution of 1:5, giving afinal sample dilution factor of 1:10,000 for unenriched samples, and 1:2,000 forenriched samples.

Measure the samplesFor all template preparation methods, measure samples using the following steps:

1. In the Guava™ Express Pro Software, open a new data set, then load the settingsthat you saved during “Set up the Guava™ easyCyte 5 Benchtop FlowCytometer“ on page 3.

2. When prompted, after loading the saved settings, place a Guava™ tubecontaining at least 200 µL Guava™ Buffer on the instrument, then press OK.

IMPORTANT! Do not adjust any of the saved settings.

3. When the instrument displays Adjust Settings at the bottom of the screen, pressNext Step. Do not save changes when prompted.

For ISPs preparedwith the Ion Chef™

System

Ion Sphere Particles (ISPs) Quality Assessment Using Guava™ easyCyte™ 5 Flow CytometerMeasure the samples


4. Enter the following information in the Sample Information panel on the left sideof the screen:

• Sample ID: Enter the sample name

• Events to Acquire: 3000

• Dilution Factor: Enter theappropriate dilution factor

• Original Volume: Enter the originalvolume of the sample from whichyou took the aliquot.

Note: Approximate original volumespresented below. Enter the actualoriginal volume if different.· Ion OneTouch™ 2 System:

· Unenriched: 0.1 mL, or 1 mL· Enriched: 0.1 mL

· Ion Chef™ System:· Unenriched: 1 mL· Enriched: 0.1 mL

5. Vortex the sample well, then immediately load it on the Guava™ easyCyte 5Benchtop Flow Cytometer. Press Acquire Sample.

6. During the run, check the sample concentration (cells/µL) shown in the panel onthe left side of the screen. If the cells/µL >1,000, abort the run, then dilute thesample to obtain a concentration of 200–1,000 cells/µL. In the SampleInformation panel, update the Dilution Factor with the new dilution beforererunning the sample.

Note: The most accurate count is obtained when the concentration is 200–1,000 cells/µL.



Example results

Example results for an unenriched Ion PI™ Ion Sphere™ Particles sample prepared on theIon OneTouch™ 2 System

Example results for an enriched Ion PI™ Ion Sphere™ Particles sample prepared on theIon OneTouch™ 2 System



Analyze the samples

IMPORTANT! Use the following metrics together to evaluate a sample, but not aspass/fail criteria.

For all template preparation methods, use Plot 2 [Green Fluorescence (GRN-HLog) v.Forward Scatter (FSC-HLog)] to estimate the percent of templated ISPs in the sampleand the total ISPs recovered:

1. Right-click Plot 2, then select Add Markers4Rectangular Marker.

2. Right-click Plot 2, then select Add Markers4Quad Stat Marker.

3. Adjust the M1 and M2 markers:

• Adjust the M1 boundaries to exclude the low-fluorescent signal population.• If there is an unwanted population with low forward scatter, adjust the M2

horizontal axis to remove this population, if needed.• Place the M2 vertical axis directly to the left of the templated ISP population.

1 2

3

1 Low-fluorescent signal population2 Non-templated ISPs3 Templated ISPs

Note: The marker settings are not identical for each sample. Adjust each settingaccordingly.

Ion Sphere Particles (ISPs) Quality Assessment Using Guava™ easyCyte™ 5 Flow CytometerAnalyze the samples


4. If you entered the correct dilution factor in the Guava™ Express Pro Software,you can determine the total number of recovered ISPs as follows:

• If the original sample volume is 1 mL: The M1 cell/mL shows the totalnumber of recovered ISPs.

• If the original sample volume is not 1 mL: Multiply the M1 cells/mL by theoriginal sample volume to calculate the total number of recovered ISPs.

Example unenrichedsample generated forsequencing on the IonProton™ System

Original unenriched sample volume: 1 mLDilution factor: 10,000 (correctly entered into the Guava™ software)Total number of ISPs = M1 Cells/mL = 5.12 billion unenriched ISPs

Example enriched samplegenerated for sequencingon the Ion Proton™ System

Original enriched sample volume: 0.1 mLDilution factor: 10,000 (correctly entered into the Guava™ software)Total number of ISPs = M1 Cells/mL x sample volume = 5.63 billion x 0.1 mL =0.563 billion enriched ISPs

5. Compare the relative fluorescence of the non-templated ISP and templated ISPpopulations.In the plot view, find the lower-left quadrant (LL) x-median value (non-templated ISP population) and the lower-right quadrant (LR) x-median value(templated ISP population).These values provide a general idea of the template load. The templatedpopulation typically has a much higher fluorescence value (higher amount ofDNA) than the non-templated population. The actual values vary from sample tosample.



6. Calculate the percentage of positive ISPs in the sample:% positive ISPs = [(LR %Total)/(M1 %Total)] × 100

Example unenrichedsample generated forsequencing on the IonProton™ System

1 2

1 % Positive ISPs = (21.20/91.30) × 100 = 23.2%2 Relative fluorescence: 27.87 to 264.95 (non-templated to templated)

Example enriched samplegenerated for sequencingon the Ion Proton™ System

1 2

1 % Positive ISPs = (91.6/94.53) × 100 = 96.9%2 Relative fluorescence: 25.52 to 88.43 (non-templated to templated)

• For unenriched samples: To produce the highest-quality sequencing data,the percent templated ISPs should be in the range of 10–25%. However,samples outside this range can still meet sequencing specifications.

• For enriched samples: For successfully enriched samples, the percenttemplated ISPs is usually >80%, however, samples outside this range can stillmeet sequencing specifications.

Note: Plot 3 is not used in the preceding calculations. If you adjust the Plot 3histogram M1 marker to cover the entire fluorescent signal population, adjust theM2 marker to cover the high fluorescent signal population, and did not excludeforward scatter in Plot 2, then the results for the Plot 3 M1 and M2 markersshould be in agreement with the Plot 2 M1 and LR markers.



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Ion Sphere Particles (ISPs) Quality Assessment Using Guava™ easyCyte™ 5 Flow CytometerCustomer and technical support



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The information in this guide is subject to change without notice.DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USEOF IT.

Revision history: Pub. No. MAN0007496Revision Date Description

B.0 24 July 2017 Updated with guidance for ISPs prepared with Ion Chef™ System, and for ISPs for sequencing on the Ion S5™ SystemA.0 15 January 2014 • Updated for Ion PI™ Template OT2 200 Kit v2 and v3

• Version numbering changed to alphanumeric format and reset to A.0 in conformance with internal documentcontrol procedures

1.0 17 January 2013 New instructions for quality assessment of Ion PI™ ISPs prepared using the Ion PI™ Template OT2 200 Kit for sequencingon the Ion Proton™ System

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