Thermo Fisher Scientific Ion AmpliSeq Microbiome Health Research Assay GX User guide

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

Ion AmpliSeq™ Microbiome Health Research

Assay GX

USER GUIDE

For use with the Genexus™ Integrated Sequencer and GX7™ Chip

Catalog NumberA47022

Publication Number MAN0026455

Revision A.0

Life Technologies Corporation | 7335 Executive Way | Frederick, Maryland 21704 USA

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

Revision history:MAN0026455 A.0 (English)

Revision Date Description

A.0 11 July 2023 Initial release.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE

LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR

ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product,

you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. Agilent and

Bioanalyzer are trademarks of Agilent Technologies, Inc. Horizon is a trademark of Horizon Discovery Group PLC. Microsoft and Excel

are trademarks of Microsoft Corp. Seraseq and SeraCare are trademarks of SeraCare Life Sciences, Inc.

Contents

About thisguide ........................................................... 6

Purpose of thisguide ............................................................ 6

Prerequisites ................................................................... 6

■CHAPTER1Productinformation .................................................. 7

Productdescription ............................................................. 7

Ion AmpliSeq™ Microbiome Health Research AssayGX .............................. 9

Recommended materials for use with the Ion AmpliSeq™ Microbiome Health

Research AssayGX .......................................................... 10

Recommended materials for nucleic acid isolation, quantiﬁcation, and

quality control ........................................................... 10

Genexus™ Controls ........................................................ 10

Required materials not supplied for use with the Genexus™ IntegratedSequencer ...... 11

Genexus™ IntegratedSequencer ............................................. 11

General laboratory supplies and reagents ..................................... 11

Genexus™ BarcodesAS .................................................... 12

Genexus™ Templating Strips 3‑GX7™ and4 ................................... 12

Genexus™ Pipette Tips ..................................................... 13

Genexus™ SequencingKit .................................................. 13

GX7™ Chip and Genexus™Coupler .......................................... 14

Genexus™ ConicalBottles .................................................. 14

Nucleic Acid to Resultworkﬂow ................................................. 15

■CHAPTER2Before youbegin .................................................... 16

Proceduralguidelines ........................................................... 16

Guidelines for nucleic acid isolation, quantiﬁcation, quality control, and inputamount .. 17

Guidelines for Genexus™ Integrated Sequencer operation ........................... 17

Guidelines for panel and reagent use andhandling ................................. 18

Guidelines for preventingcontamination .......................................... 19

Before each use of thekit ....................................................... 20

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 3

Precautions ................................................................... 21

Avoid nucleic acidcontamination ............................................ 21

Avoid chipdamage ........................................................ 21

Avoid strong electromagnetic radiation ....................................... 21

Protection byequipment .................................................... 21

Install software and assay deﬁnition ﬁle updates ................................... 21

■CHAPTER3Plan and manageruns ............................................. 23

Types of Ion AmpliSeq™ Microbiome Health Research Assay GX runs ................ 23

System-installed assays for use with the Ion AmpliSeq™ Microbiome Health

Research AssayGX .......................................................... 24

Enter samples in the Genexus™ Software ......................................... 24

Plan a Nucleic Acid to Resultrun ................................................. 25

Chipcapacity .................................................................. 27

■CHAPTER4Prepare the sample input plate .................................... 28

Guidelines for DNA isolation, quantiﬁcation, andinput .............................. 28

Quantify nucleic acidsamples ................................................... 28

Dilute the samples (if needed) and load the sample plate ............................ 29

■CHAPTER5Load the sequencer and start arun ............................... 31

Before youbegin ............................................................... 31

Centrifuge library and templating reagent strips using the Genexus™ Strip

Centrifuge Adapter ...................................................... 34

Load the sequencer and start arun .............................................. 35

Clear the instrument deck and perform a UVClean ................................. 43

■CHAPTER6Review data and results ............................................ 46

Results navigation bar overview ................................................. 46

View sample results ............................................................ 47

Microbiome assay results ................................................... 49

View the QC results ........................................................ 60

View keyﬁndings .......................................................... 62

Compare results from multiple samples or sampleruns ......................... 63

Review run results ............................................................. 71

View assay metrics and the run report ........................................ 72

View the run summary ...................................................... 76

Assign PCR Plate .......................................................... 77

Contents

4Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

■APPENDIXATroubleshooting .................................................... 78

Ion AmpliSeq™ Microbiome Health Research AssayGX ............................. 78

Genexus™ Integrated Sequencer—general and QC troubleshooting .................. 79

Genexus™ Software ............................................................ 80

■APPENDIXBSupplementalinformation ........................................ 81

Plan a single panel Nucleic Acid to Resultrun ..................................... 81

Copy/edit the Microbiome assay to sequence with either panel individually ....... 82

Chipcapacity .................................................................. 83

■APPENDIXCSafety ............................................................... 84

Biological hazardsafety ......................................................... 84

Chemicalsafety ................................................................ 85

■Documentation and support ....................................................... 86

Relateddocumentation ......................................................... 86

Customer and technical support ................................................. 87

Contents

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 5

About this guide

IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix

in this document.

Purpose of this guide

This user guide describes how to use the Ion AmpliSeq™ Microbiome Health Research Assay GX to

perform Nucleic Acid to Result next-generation sequencing (NGS) runs on the Genexus™ Integrated

Sequencer. The guide provides experienced-user procedures for run planning in Genexus™ Software,

loading samples onto the sequencer, starting an instrument run, and data analysis in Genexus™

Software.

If you are a new user, or you need detailed descriptions of all features of the Genexus™ Integrated

Sequencer, and Genexus™ Software, see the following resources available at thermoﬁsher.com.

•Genexus™ Integrated Sequencer User Guide (Pub.No.MAN0017910)

•Genexus™ Software 6.8 User Guide (Pub.No.MAN0026409) or Genexus™ Software 6.8 Help

(available in the software)

Prerequisites

Category Prerequisites

Instrument •Access to the Genexus™ Integrated Sequencer

•Stock of reagents and supplies that are compatible with the Ion AmpliSeq™

Microbiome Health Research Assay GX and the Genexus™ Integrated Sequencer

For ordering information, see “Required materials not supplied for use with the Genexus™

Integrated Sequencer” on page11.

Software •Genexus™ Software 6.8 or later

Functional knowledge

and understanding

•Key steps in a next-generation sequencing (NGS) workﬂow

•Main functions of the Genexus™ Integrated Sequencer

•Main features of the Genexus™ Software 6.8 or later

6Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

Product information

■ Productdescription .................................................................... 7

■ Ion AmpliSeq™ Microbiome Health Research AssayGX .................................... 9

■ Recommended materials for use with the Ion AmpliSeq™ Microbiome Health Research

AssayGX ............................................................................ 10

■ Required materials not supplied for use with the Genexus™ IntegratedSequencer ............ 11

■ Nucleic Acid to Resultworkﬂow ........................................................ 15

Product description

The Ion AmpliSeq™ Microbiome Health Research Assay GX is a comprehensive, targeted next-

generation sequencing (NGS) assay for characterizing the human gut microbiome. The assay consists

of two pools—the Ion AmpliSeq™ Microbiome Health Research – 16S rRNA Gene Pool and the Ion

AmpliSeq™ Microbiome Health Research – Target Species Pool—of Ion AmpliSeq™ oligonucleotide

primers and associated reagents to generate amplicon libraries. Using the kit, microbial DNA isolated

from human stool samples is used to generate libraries for templating and sequencing on the Ion

Torrent™ Genexus™ Integrated Sequencer System.

The 16S rRNA Gene Pool contains primers that provides high coverage of the 16S ribosomal RNA

(rRNA) gene, by targeting 8 out of 9 hypervariable regions present in the 16S rRNA gene. As the 16S

rRNA gene is present in every bacteria, this pool detects and quantiﬁes all microbes of bacterial origin

in the sample.

The Target Species Pool contains primers targeting 73 key bacterial species involved in regulating

human gut health. The Target Species Pool provides accurate species-level resolution for a subset of

gut bacteria and supplements detection by the16S rRNA gene, which due to its inherent homology is

sometimes unable to resolve bacterial identity at the species-level.

The kit includes the following key features:

•Up to 48 samples can be sequenced on a single GX7™ Chip across two runs. 24 samples in 2lanes

per run.

•Typically, the entire Nucleic Acid to Result workﬂow can be completed in about 29hours when

running 24 samples (2lanes), including the analysis. When running 12 samples (1lane) only, the full

workﬂow takes about 20hours.

•The recommended DNA sample input concentration is 0.11ng/μL for the default assay or custom

assays containing both the Ion AmpliSeq™ Microbiome Health Research – 16S rRNA Gene Pool

and the Ion AmpliSeq™ Microbiome Health Research – Target Species Pool.

1

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 7

•For custom assays containing only a single pool.

–The recommended DNA sample input concentration is 0.11ng/μL for the Ion AmpliSeq™

Microbiome Health Research – 16S rRNA Gene Pool.

–The recommended DNA sample input concentration is 0.56ng/μL for the Ion AmpliSeq™

Microbiome Health Research – Target Species Pool.

IMPORTANT! Ion AmpliSeq™ Microbiome Health Research Assay GX is only supported for use with

the GX7™ Chip and Genexus™ Barcodes 1–48 AS (Cat.No.A54129) or Genexus™ Barcodes 49–96 AS

(Cat.No.A54130). The product is not supported for use with the GX5™ Chip or Genexus™ Barcodes

1–32AS, 33–64AS, and 65–96AS.

Chapter1Product information

Product description

1

8Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

Ion AmpliSeq™ Microbiome Health Research Assay GX

The Ion AmpliSeq™ Microbiome Health Research Assay GX (Cat.No.A47022) includes the Ion

AmpliSeq™ Microbiome Health Research – 16S rRNA Gene Pool and Ion AmpliSeq™ Microbiome

Health Research – Target Species Pool for stool microbiome samples, and the Genexus™ Strip1 and

Genexus™ Strip2‑AS. The contents of each kit are sucient for sequencing up to 24 stool samples.

Each primer pool in the panel is provided in pairs of tubes, where each tube pair contains one tube with

primers in position 1 and one empty uncapped tube in position 2. Two 8‑strip packs of the Genexus™

Strip1 and Genexus™ Strip2‑AS (24 total of each strip) are provided with each kit. If needed, the

Genexus™ Library Strips 1 and 2‑AS combo kit (Cat.No.A40252) can be ordered separately.

Ion AmpliSeq™ Microbiome Health Research Assay GX (Cat.No.A47022)

Contents Carrier color Number

of reactions Amount PartNo. Storage

Ion AmpliSeq™ Microbiome Health Research Assay GX pools

Ion AmpliSeq™ Microbiome Health Research

– 16S rRNA Gene Pool (position 1)

Magenta 24

(6 reactions

/tube)

4tubes A47023 −30°C to −10°C

Ion AmpliSeq™ Microbiome Health Research

– Target Species Pool (position 1)

Pale green 24

(6 reactions

/tube)

4tubes

Genexus™ Library Strips 1 and 2‑AS (Cat.No.A40252)

Genexus™ Strip1 Light red 48

(6 single‑pool

reactions/strip)

1×8strips A46812 2°C to 8°C

Genexus™ Strip2‑AS Light blue 48

(6 single‑pool

reactions/strip)

1×8strips A46813 −30°C to −10°C

Chapter1Product information

Ion AmpliSeq™ Microbiome Health Research Assay GX 1

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 9

Recommended materials for use with the Ion AmpliSeq™

Microbiome Health Research Assay GX

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that

the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Recommended materials for nucleic acid isolation, quantiﬁcation, and

quality control

Item Source

Nucleic acid isolation

KingFisher™ Puriﬁcation System thermoﬁsher.com/kingﬁsher

MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit A42358 (tubes)

A42357 (plates)

Nucleic acid quantiﬁcation

Qubit™ 4 Fluorometer[1] and one or more of the following kits:

•Qubit™ dsDNA HS Assay Kit

•Qubit™ dsDNA BR Assay Kit

Q33238

Q32851, Q32854

Q32853, Q32850

Recommended controls

Gut Microbiome Genomic Mix MSA-1002

MSA-1006

American Type Culture Collection (ATCC)

Nuclease-Free Water (not DEPC-Treated) AM9930

(or equivalent)

[1] Qubit™ 2.0 Fluorometer and later are supported.

Genexus™ Controls

The Ion Torrent™ Genexus™ Controls kit (Cat. No. A40267) provides sucient Genexus™ Control

Library‑AS to perform four Library to Result runs. The kit also provides sucient Genexus™ Control

Panel‑AS and Genexus™ DNA Control to perform eight Nucleic Acid to Result runs.

Note: The Genexus™ Control Library‑AS is barcoded with IonCode™ 0101.

Component Quantity Storage

Genexus™ Control Library‑AS 1 tube

−30°C to −10°CGenexus™ Control Panel‑AS 8 carriers (white)

Genexus™ DNA Control 2 tubes

Chapter1Product information

Recommended materials for use with the Ion AmpliSeq™ Microbiome Health Research Assay GX

1

10 Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

Required materials not supplied for use with the Genexus™

Integrated Sequencer

Genexus™ Integrated Sequencer reagents and supplies can be ordered as kits and starter packs. In

addition, most consumables can also be ordered individually. The following tables provide information

about the various ordering options.

Note: Consumables that have catalog numbers are orderable. Components that have part numbers

cannot be ordered individually.

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates

that the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier. Catalog

numbers that appear as links open the web pages for those products.

Genexus™ Integrated Sequencer

Components Cat. No.

Genexus™ Integrated Sequencer A45727

General laboratory supplies and reagents

Item Source

Sorvall™ ST 8 Small Benchtop Centrifuge[1] (or equivalent), with

Thermo Scientiﬁc™ M10 Microplate Swinging Bucket Rotor (or equivalent), and

Sealed Bucket; Capacity: 4 Standard or 2 Midi-Deepwell plates (Set of2) (or equivalent)

75007200

75005706

75005721

Microcentrifuge[2] MLS

MicroAmp™ EnduraPlate™ Optical 96-Well Clear Reaction Plates with Barcode 4483352

4483354

Adhesive PCR Plate Foils AB0626

2‑, 20‑, 200‑, and 1,000‑µL pipettes and appropriate ﬁltered tips MLS

Nuclease‑free microcentrifuge tubes, 1.5-mL or 1.7-mL MLS

Vortex mixer with a rubber platform MLS

Gloves, powder-free nitrile MLS

Ice buckets and ice —

Nuclease-free water, molecular biology grade MLS

Isopropyl alcohol, 70% solution MLS

Chapter1Product information

Required materials not supplied for use with the Genexus™ Integrated Sequencer 1

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 11

(continued)

Item Source

Wipes, disposable lint-free MLS

(Optional) Uninterruptible Power Supply (UPS)[3] MLS

[1] For centrifuging library and templating reagent strips using the Genexus™ Strip Centrifuge Adapter.

[2] Must fit standard 0.2- and 1.5‑mL microcentrifuge tubes and generate 15,000 × g. To convert the RPMs of your centrifuge to RCF in

units of gravity, see tools.thermofisher.com/content/sfs/brochures/TR0040-Centrifuge-speed.pdf.

[3] For laboratories that experience frequent power outages or line voltage fluctuations, we recommend that you use an uninterruptible

power supply that is compatible with 2500W output or higher.

Genexus™ Barcodes AS

Ion Torrent™ Genexus™ Barcodes AS are supplied in plates containing 48 dual barcodes per plate.

Item Label color Cat. No. Quantity Storage

Genexus™ Barcodes 1–48 AS Blue A54129 1 plate

15°C to 30°C

Genexus™ Barcodes 49–96 AS Blue A54130 1 plate

Note: Genexus™ Barcodes 1–32AS (Cat.No.A40258), Genexus™ Barcodes 33–64AS

(Cat.No.A40259), and Genexus™ Barcodes 65–96AS (Cat.No.A40260) are not supported for use

with the Ion AmpliSeq™ Microbiome Health Research Assay GX.

Genexus™ Templating Strips 3‑GX7™ and 4

Ion Torrent™ Genexus™ Templating Strips 3‑GX7™ and 4 (Cat. No. A46817) are ordered as kits with

8pairs of strips per kit.

Component Carrier color Part No. Quantity per kit Storage

Genexus™ Strip3‑GX7™Green A46818 8 strips 2°C to 8°C

Genexus™ Strip4 Yellow A46816 8 strips −30°C to −10°C

Chapter1Product information

Required materials not supplied for use with the Genexus™ Integrated Sequencer

1

12 Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

Genexus™ Pipette Tips

Ion Torrent™ Genexus™ Pipette Tips (Cat.No.A40266) are ordered in packs of 12 racks each. The

number of pipette tip racks that are required for your experiment depends on the number of reactions

and the sample type. For the Ion AmpliSeq™ Microbiome Health Research Assay GX, 4 racks are

sucient for sequencing up to 4 samples and no-template controls on a single GX7™ Chip lane.

Item Cat. No. Quantity Storage

Genexus™ Pipette Tips A40266 12 racks 15°C to 30°C

Genexus™ Sequencing Kit

The Ion Torrent™ Genexus™ Sequencing Kit (Cat. No. A40271) provides reagents and solutions sucient

to sequence up to 2full chips.

Component Part No. Quantity Storage

Genexus™ Cartridge A40272 2 cartridges −30°C to −10°C

Genexus™ Bottle 2 A40273 4bottles 15°C to 30°C

Genexus™ Bottles 1 and 3 A40274 2bottles each

(4 bottles total)

Chapter1Product information

Required materials not supplied for use with the Genexus™ Integrated Sequencer 1

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 13

GX7™ Chip and Genexus™ Coupler

The GX7™ Chip and Genexus™ Coupler (Cat. No. A46731) are ordered as a set that contains 2 chips

and 2 couplers, sucient for up to 8 sequencing runs.

Component Part No. Quantity Storage

GX7™ Chip

7

100093568 2 chips 15°C to 30°C

Genexus™ Coupler 100081252 2 couplers

Genexus™ Conical Bottles

Genexus™ Conical Bottles (Cat. No. A40275) are installed in the sequencing reagents bay and serve as

reservoirs for nucleotide reagent dilutions. For information on when and how to replace the bottles, see

the Genexus™ Integrated Sequencer User Guide (Pub. No. MAN0017910). .

Component Quantity Storage

Genexus™ Conical Bottles 5 bottles 15°C to 30°C

Chapter1Product information

Required materials not supplied for use with the Genexus™ Integrated Sequencer

1

14 Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

Nucleic Acid to Result workﬂow

The Nucleic Acid to Result workﬂow starts from puriﬁed and quantiﬁed nucleic acid samples. Puriﬁed

nucleic acid samples are pipet into a 96-well sample input plate and then loaded into the Genexus™

Integrated Sequencer for library preparation, templating, and sequencing.

Nucleic Acid to Result

Prepare puriﬁed DNA

We recommend the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit for

extraction of DNA from stool samples for use in Nucleic Acid to Result runs.

Use a Qubit™ Fluorometer to accurately measure sample concentration.

Note: A KingFisher™ puriﬁcation system can be used to automate extraction of

DNA from stool samples.

up to

3hr

Enter samples (page24)

Before you plan a run in Genexus™ Software, you must enter sample

information in the software to assign sample names and other information.

10 min

Plan a Nucleic Acid to Result run (page25)

In the Genexus™ Software, plan a Nucleic Acid to Result run to create a

sequencing run that starts with nucleic acid samples. The run plan deﬁnes the

samples, assays, sequencing reagents, and the number of sequencing chip

lanes that are needed.

5 min

Prepare sample input plate (page28)

Transfer puriﬁed DNA to a 96-well sample input plate for loading into the

Genexus™ Integrated Sequencer. Add positive and negative controls to the

sample input plate if included in the run plan.

10 min

Sequence the puriﬁed nucleic acid sample on the

Genexus™ Integrated Sequencer (page35)

Load the input plate, and all reagents and consumables needed for the run,

onto the integrated sequencer. Select the Nucleic Acid to Result run plan, then

press start.

up to

24hr

Chapter1Product information

Nucleic Acid to Result workﬂow 1

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 15

Before you begin

■ Proceduralguidelines ................................................................. 16

■ Guidelines for nucleic acid isolation, quantiﬁcation, quality control, and inputamount ......... 17

■ Guidelines for Genexus™ Integrated Sequencer operation ................................. 17

■ Guidelines for panel and reagent use andhandling ....................................... 18

■ Guidelines for preventingcontamination ................................................. 19

■ Before each use of thekit ............................................................. 20

■ Precautions .......................................................................... 21

■ Install software and assay deﬁnition ﬁle updates .......................................... 21

Procedural guidelines

•Use only the reagents and supplies that have been validated for the Ion AmpliSeq™ Microbiome

Health Research Assay GX. For a list of validated reagents and supplies, see “Required materials

not supplied for use with the Genexus™ Integrated Sequencer” on page11 and “Recommended

materials for use with the Ion AmpliSeq™ Microbiome Health Research Assay GX” on page10.

•Use good laboratory practices to minimize cross-contamination of products. Keep all tubes sealed

until immediately before loading onto the Genexus™ Integrated Sequencer.

•Use controls to identify or rule out reagent contamination. For a list of validated controls, see

“Genexus™ Controls” on page10.

•Each GX7™ Chip can accommodate up to 48 barcoded microbiome libraries, per 2 lanes per run.

Either 24 samples if using both pools or 48 samples if only using a single pool.

•Minimize freeze‑thaw cycles of the Ion AmpliSeq™ Microbiome Health Research – 16S rRNA Gene

Pool, Ion AmpliSeq™ Microbiome Health Research – Target Species Pool tubes. Thaw only the

number of panel tubes that are required for a given experiment and keep the thawed panels at 4°C

until ready to use. Store unused panels at −30°C to −10°C.

Note: One set of panels (Ion AmpliSeq™ Microbiome Health Research – 16S rRNA Gene Pool, Ion

AmpliSeq™ Microbiome Health Research – Target Species Pool tubes) is sucient for processing

up to 4samples.

•Do NOT store the Ion AmpliSeq™ Microbiome Health Research – 16S rRNA Gene Pool, Ion

AmpliSeq™ Microbiome Health Research – Target Species Pool tubes, and the Genexus™ Strip1

and Genexus™ Strip2‑AS on the Genexus™ Integrated Sequencer for more than 24 hours before

starting an instrument run.

2

16 Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

Guidelines for nucleic acid isolation, quantiﬁcation, quality

control, and input amount

These are general guidelines for manual isolation and quantiﬁcation of DNA for Nucleic Acid to Result

runs. If you do not dilute sample concentrations manually to the target concentration of the assay,

quantify sample nucleic acid concentrations ahead of time so concentrations are available to enter

during run planning.

•For validated kits to use for isolation, quantiﬁcation, and quality control of nucleic acid samples,

see “Recommended materials for nucleic acid isolation, quantiﬁcation, and quality control” on

page10.

•Quantiﬁcation methods such as densitometry (for example, using a NanoDrop™ spectrophotometer)

are not recommended, because they are not speciﬁc for DNA. Use of these methods can lead

to gross overestimation of the concentration of sample nucleic acid, under-seeding of the target

ampliﬁcation reaction, and low library yields.

•The recommended DNA sample input concentration is 0.11ng/μL for the Ion AmpliSeq™

Microbiome Health Research – 16S rRNA Gene Pool and 0.56ng/μL for the Ion AmpliSeq™

Microbiome Health Research – Target Species Pool.

Guidelines for Genexus™ Integrated Sequencer operation

•Follow guidance that is provided by Genexus™ Software when you plan a run to determine which

consumables must be loaded and which consumables can be reused from a previous run.

•Follow guidance that is provided by the software when you plan a run to determine how many

samples can be run with a given assay or assays in an instrument run. The number of samples that

can be included in a sequencing run depends on multiple factors.

Limiting factor Description

The number of available barcodes

in the barcode plate

The maximum number of available barcodes per run is 48.

Each sample requires 2 barcodes when running both 16S and

target species primer pools.

Maximum number of target

ampliﬁcation reactions per run

One library strip pair has the reagents necessary for 6 target

ampliﬁcation reactions. With a maximum of 8 library strip pairs

loaded, a maximum of 24samples can be run using the Ion

AmpliSeq™ Microbiome Health Research Assay GX, which contains

2primer pools.

The number of primer pools per

assay

The number of samples in a run multiplied by the total number of

primer pools in the assays that are used in a run cannot exceed 48.

The number of unused lanes on

an installed chip

A maximum of 4 lanes are available on a single GX7™ Chip.

The minimum read count per

sample for an assay

The minimum read count per sample parameter is set during assay

creation.

•One library strip pair is needed for each primer tube position 1–8 that is ﬁlled in a run.

•One template strip pair is needed for each chip lane that is used in a run.

Chapter2Before you begin

Guidelines for nucleic acid isolation, quantiﬁcation, quality control, and input amount 2

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 17

•Consumables are conﬁgured to support sample batch sizes in multiples of 4-6 samples. The most

ecient use of consumables occurs when samples are run in multiples of four or six.

•Each GX7™ Chip can accommodate up to 48 microbiome DNA samples for analysis, 24 samples if

running both pools, across 2lanes per run.

•If a chip installed in a sequencer has unused lanes, do not remove it unless you are sure that

you want to replace it with a new chip. After a partially used chip has been removed from the

sequencer, it cannot be reinserted and reused. The sequencer cannot track lane usage after chip

removal.

•You can remove a chip in one of the following situations.

–After all the lanes of a chip are used in a run, the chip shuttles to the install position and you

are asked to remove the used chip.

–When you select a run plan that requires more lanes than are available on the installed chip,

you are asked to remove the partially used chip, and the sequencer performs a post-chip

clean. In addition, you must clear consumables from the lower sequencing reagents bay, even

if only a single lane of the chip was used.

•The Genexus™ Integrated Sequencer can track used and unused barcodes in barcode plates in

Genexus™ Software 6.2.0 and later, enabling you to swap plates between runs if needed, and

reload a partially used barcode plate for a run if a sucient number of barcodes are available on

the plate.

•After loading in the sequencer, reusable consumables, such as chips and sequencing reagents bay

components, must be used within 14 days for optimal results.

Guidelines for panel and reagent use and handling

•Use only the reagents and supplies that have been recommended in “General laboratory supplies

and reagents” on page11 and “Recommended materials for nucleic acid isolation, quantiﬁcation,

and quality control” on page10.

•Keep panel tubes capped until immediately before loading in the Genexus™ Integrated Sequencer.

•If using, thaw positive controls on ice for 30 minutes. After the positive controls are completely

thawed, vortex the tubes, then centrifuge to collect tube contents. Return to ice before loading into

sample plate.

IMPORTANT! Ensure that contents of the control tubes are completely thawed before adding to

the sample plate.

•Equilibrate Genexus™ Strip1 and Genexus™ Strip3‑GX5™ (or Genexus™ Strip3‑GX7™) at room

temperature for 30 minutes before loading in the sequencer.

•Thaw Genexus™ Strip2‑AS (or Genexus™ Strip2‑HD) and Genexus™ Strip4 on ice for 30 minutes.

IMPORTANT! Ensure that strip contents are completely thawed before loading in the sequencer.

•Thawed library and templating strips can be vortexed on a platform vortexer to dissolve precipitate

or dislodge air bubbles. If you vortex, you must centrifuge the strips to collect tube contents using

the Genexus™ Strip Centrifuge Adapter to hold strips during centrifugation.

For information on obtaining and using the Genexus™ Strip Centrifuge Adapter, see “Centrifuge

library and templating reagent strips using the Genexus™ Strip Centrifuge Adapter” on page34.

Chapter2Before you begin

Guidelines for panel and reagent use and handling

2

18 Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

•Keep thawed panel, Genexus™ Strip2‑AS (or Genexus™ Strip2‑HD), Genexus™ Strip4, and the

sample plate on ice or at 4°C until ready to load in the sequencer.

•Do not freeze‑thaw the panel. Thaw only the number of panel tubes that are required for an

instrument run. Discard unused panel tubes after they are thawed. Store panel tubes at −30°C to

−10°C.

•If you are using an assay that uses Ion AmpliSeq™ HD chemistry, do not combine the contents of

panel tubes. Forward and reverse primers must remain separate, until they are combined by the

sequencer as part of the on‑instrument library preparation workﬂow.

•Do not store primer pool tubes or reagent strips on the sequencer for more than 24 hours before

starting an instrument run.

Guidelines for preventing contamination

We recommend following these guidelines to prevent cross-contamination of samples and controls

between and within sequencing runs.

Personal protective equipment

•Wear a lab coat that is reserved for sequencing work and is laundered frequently. If possible,

change to a fresh lab coat before setting up a run, or use new sleeve covers.

•Wear fresh gloves to load the instruments, including during the loading of the sequencing chip and

coupler. Do not remove gloves to install the sequencing chip.

•If you are using positive controls and a no-template control (NTC) in the run, change gloves

between dispensing these controls, and if applicable, between dispensing samples and controls in

sample plate wells.

Equipment and instrument cleaning

•If you are preparing samples and sample plates in a hood (recommended), illuminate the hood with

UV light for 15 minutes before use.

•Before use, wipe working surfaces of the hood or bench where samples or sample plates are

handled, and other equipment such as vortexers, microcentrifuges, and pipettors with lint-free

wipes moistened with DNAZap™ decontamination solutions. Wipe with solution1 ﬁrst, then follow

with solution 2. Alternatively, a 10% solution of commercial bleach can be used. Follow with wiping

of bench and equipment surfaces with wipes moistened with 70% isopropanol or 70% ethanol.

•Before and after a run, sequentially wipe instrument deck surfaces with lint-free wipes moistened

with the two DNAZap™ decontamination solutions. Follow with wiping of deck surfaces with wipes

moistened with 70% isopropanol or 70% ethanol. The robotic pipettor arm can also be cleaned in

this manner.

IMPORTANT! Do not spray decontamination solution or alcohol solution directly onto deck

surfaces or into deck openings. Instead, use a lint-free wipe moistened with solution to clean

surfaces. Do not use bleach to clean instrument surfaces.

•We recommend that you centrifuge the sample plate after sealing. Ensure that the centrifuge has

been wiped down and cleaned before centrifuging the plate.

Chapter2Before you begin

Guidelines for preventing contamination 2

Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide 19

Workﬂow tips

•After dispensing a positive control or sample (if applicable) in a sample plate well, do not pass

the end of the used tip over wells intended for other samples or NTC. This practice minimizes the

chance of depositing microdroplets in adjacent wells.

•If you dilute samples on the sample plate and vortex the plate after sealing, apply the foil seal

carefully between wells with an applicator before vortexing to ensure that the seal is complete and

contamination between wells does not occur.

•If possible, have a pipettor and tips reserved for dispensing only NTC.

•Before disposal, close or cap used sample and positive control tubes to avoid creation of aerosols.

•Avoid touching the foil seals of the reagent strips, barcode plate, and sample plate.

•When loading the sequencer deck for a run, install the sample plate last.

•After a run, seal the PCR ampliﬁcation plate with a foil seal before removing the plate from the PCR

ampliﬁcation station. Sealing the plate before removal helps prevent contamination of libraries if

libraries are recovered for reuse.

Before each use of the kit

Thaw assay reagents as indicated:

•Thaw Ion AmpliSeq™ Microbiome Health Research – 16S rRNA Gene Pool, Ion AmpliSeq™

Microbiome Health Research – Target Species Pool on ice or at 4°C for at least 30 minutes and

keep on ice until immediately before loading onto the Genexus™ Integrated Sequencer.

•Equilibrate Genexus™ Strip1 and Genexus™ Strip3‑GX7™ at room temperature for at least

30 minutes before loading onto the Genexus™ Integrated Sequencer.

•Thaw Genexus™ Strip2‑AS and Genexus™ Strip4 on ice or at 4°C for at least 30 minutes and keep

on ice until immediately before loading onto the Genexus™ Integrated Sequencer.

IMPORTANT! Ensure that the strip contents are completely thawed before installing in the sequencer.

When thawed, gently tap tubes and strips on the benchtop to remove any bubbles and collect the

contents at the bottom of each tube. For information about preparing all reagents and consumables for

loading onto the instrument, see “Before you begin” on page31.

Note: If tapping fails to dislodge a bubble, you can dislodge large bubbles using the technique that

is described in “Centrifuge library and templating reagent strips using the Genexus™ Strip Centrifuge

Adapter” on page34.

Chapter2Before you begin

Before each use of the kit

2

20 Ion AmpliSeq™ Microbiome Health Research Assay GX User Guide

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Thermo Fisher Scientific Ion AmpliSeq Microbiome Health Research Assay GX User guide

Thermo Fisher Scientific Ion AmpliSeq Microbiome Health Research Assay GX User guide

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