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For Research Use Only. Not for use in diagnostic procedures.
Ion AmpliSeqā¢ Microbiome Health Research
Assay GX
USER GUIDE
For use with the Genexusā¢ Integrated Sequencer and GX7ā¢ Chip
Catalog NumberīīA47022
Publication Numberīī MAN0026455
Revisionīī A.0
Life Technologies Corporation | 7335 Executive Way | Frederick, Maryland 21704 USA
For descriptions of symbols on product labels or product documents, go to thermoļ¬sher.com/symbols-deļ¬nition.
Revision history:īīMAN0026455 A.0 (English)
Revision Date Description
A.0 11 July 2023 Initial release.
The information in this guide is subject to change without notice.
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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 Scientiļ¬c and its subsidiaries unless otherwise speciļ¬ed. Agilent and
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are trademarks of Microsoft Corp. Seraseq and SeraCare are trademarks of SeraCare Life Sciences, Inc.
Ā©2023 Thermo Fisher Scientiļ¬c Inc. All rights reserved.
Contents
About thisīguide ........................................................... 6
Purpose of thisīguide ............................................................ 6
Prerequisites ................................................................... 6
ā CHAPTERī1īīīProductīinformation .................................................. 7
Productīdescription ............................................................. 7
Ion AmpliSeqā¢ Microbiome Health Research AssayīGX .............................. 9
Recommended materials for use with the Ion AmpliSeqā¢ Microbiome Health
Research AssayīGX .......................................................... 10
Recommended materials for nucleic acid isolation, quantiļ¬cation, and
quality control ........................................................... 10
Genexusā¢ Controls ........................................................ 10
Required materials not supplied for use with the Genexusā¢ IntegratedīSequencer ...... 11
Genexusā¢ IntegratedīSequencer ............................................. 11
General laboratory supplies and reagents ..................................... 11
Genexusā¢ BarcodesīAS .................................................... 12
Genexusā¢ Templating Strips 3āGX7ā¢ andī4 ................................... 12
Genexusā¢ Pipette Tips ..................................................... 13
Genexusā¢ SequencingīKit .................................................. 13
GX7ā¢ Chip and Genexusā¢īCoupler .......................................... 14
Genexusā¢ ConicalīBottles .................................................. 14
Nucleic Acid to Resultīworkļ¬ow ................................................. 15
ā CHAPTERī2īīīBefore youībegin .................................................... 16
Proceduralīguidelines ........................................................... 16
Guidelines for nucleic acid isolation, quantiļ¬cation, quality control, and inputīamount .. 17
Guidelines for Genexusā¢ Integrated Sequencer operation ........................... 17
Guidelines for panel and reagent use andīhandling ................................. 18
Guidelines for preventingīcontamination .......................................... 19
Before each use of theīkit ....................................................... 20
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 3
Precautions ................................................................... 21
Avoid nucleic acidīcontamination ............................................ 21
Avoid chipīdamage ........................................................ 21
Avoid strong electromagnetic radiation ....................................... 21
Protection byīequipment .................................................... 21
Install software and assay deļ¬nition ļ¬le updates ................................... 21
ā CHAPTERī3īīīPlan and manageīruns ............................................. 23
Types of Ion AmpliSeqā¢ Microbiome Health Research Assay GX runsī ................ 23
System-installed assays for use with the Ion AmpliSeqā¢ Microbiome Health
Research AssayīGX .......................................................... 24
Enter samples in the Genexusā¢ Software ......................................... 24
Plan a Nucleic Acid to Resultīrun ................................................. 25
Chipīcapacity .................................................................. 27
ā CHAPTERī4īīīPrepare the sample input plate .................................... 28
Guidelines for DNA isolation, quantiļ¬cation, andīinput .............................. 28
Quantify nucleic acidīsamples ................................................... 28
Dilute the samples (if needed) and load the sample plate ............................ 29
ā CHAPTERī5īīīLoad the sequencer and start aīrun ............................... 31
Before youībegin ............................................................... 31
Centrifuge library and templating reagent strips using the Genexusā¢ Strip
Centrifuge Adapter ...................................................... 34
Load the sequencer and start aīrun .............................................. 35
Clear the instrument deck and perform a UVīClean ................................. 43
ā CHAPTERī6īīīReview data and results ............................................ 46
Results navigation bar overview ................................................. 46
View sample results ............................................................ 47
Microbiome assay results ................................................... 49
View the QC results ........................................................ 60
View keyīļ¬ndings .......................................................... 62
Compare results from multiple samples or sampleīruns ......................... 63
Review run results ............................................................. 71
View assay metrics and the run report ........................................ 72
View the run summary ...................................................... 76
Assign PCR Plate .......................................................... 77
Contents
4Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
ā APPENDIXīAīīīTroubleshooting .................................................... 78
Ion AmpliSeqā¢ Microbiome Health Research AssayīGX ............................. 78
Genexusā¢ Integrated Sequencerāgeneral and QC troubleshooting .................. 79
Genexusā¢ Software ............................................................ 80
ā APPENDIXīBīīīSupplementalīinformation ........................................ 81
Plan a single panel Nucleic Acid to Resultīrun ..................................... 81
Copy/edit the Microbiome assay to sequence with either panel individuallyī ....... 82
Chipīcapacity .................................................................. 83
ā APPENDIXīCīīīSafety ............................................................... 84
Biological hazardīsafety ......................................................... 84
Chemicalīsafety ................................................................ 85
ā Documentation and support ....................................................... 86
Relatedīdocumentation ......................................................... 86
Customer and technical support ................................................. 87
Contents
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 5
About this guide
IMPORTANT! Before using this product, read and understand the information in the āSafetyā appendix
in this document.
Purpose of this guide
This user guide describes how to use the Ion AmpliSeqā¢ Microbiome Health Research Assay GX to
perform Nucleic Acid to Result next-generation sequencing (NGS) runs on the Genexusā¢ Integrated
Sequencer. The guide provides experienced-user procedures for run planning in Genexusā¢ Software,
loading samples onto the sequencer, starting an instrument run, and data analysis in Genexusā¢
Software.
If you are a new user, or you need detailed descriptions of all features of the Genexusā¢ Integrated
Sequencer, and Genexusā¢ Software, see the following resources available at thermoļ¬sher.com.
ā¢Genexusā¢ Integrated Sequencer User Guide (Pub.īNo.īMAN0017910)
ā¢Genexusā¢ Software 6.8 User Guide (Pub.īNo.īMAN0026409) or Genexusā¢ Software 6.8 Help
(available in the software)
Prerequisites
Category Prerequisites
Instrument ā¢Access to the Genexusā¢ Integrated Sequencer
ā¢Stock of reagents and supplies that are compatible with the Ion AmpliSeqā¢
Microbiome Health Research Assay GX and the Genexusā¢ Integrated Sequencer
For ordering information, see āRequired materials not supplied for use with the Genexusā¢
Integrated Sequencerā on pageī11.
Software ā¢Genexusā¢ Software 6.8 or later
Functional knowledge
and understanding
ā¢Key steps in a next-generation sequencing (NGS) workļ¬ow
ā¢Main functions of the Genexusā¢ Integrated Sequencer
ā¢Main features of the Genexusā¢ Software 6.8 or later
6Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
Product information
ā Productīdescription .................................................................... 7
ā Ion AmpliSeqā¢ Microbiome Health Research AssayīGX .................................... 9
ā Recommended materials for use with the Ion AmpliSeqā¢ Microbiome Health Research
AssayīGX ............................................................................ 10
ā Required materials not supplied for use with the Genexusā¢ IntegratedīSequencer ............ 11
ā Nucleic Acid to Resultīworkļ¬ow ........................................................ 15
Product description
The Ion AmpliSeqā¢ 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 poolsāthe Ion AmpliSeqā¢ Microbiome Health Research ā 16S rRNA Gene Pool and the Ion
AmpliSeqā¢ Microbiome Health Research ā Target Species Poolāof Ion AmpliSeqā¢ 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
Torrentā¢ Genexusā¢ 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 quantiļ¬es 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:
ā¢Up to 48 samples can be sequenced on a single GX7ā¢ Chip across two runs. 24 samples in 2īlanes
per run.
ā¢Typically, the entire Nucleic Acid to Result workļ¬ow can be completed in about 29īhours when
running 24 samples (2īlanes), including the analysis. When running 12 samples (1īlane) only, the full
workļ¬ow takes about 20īhours.
ā¢The recommended DNA sample input concentration is 0.11īng/Ī¼L for the default assay or custom
assays containing both the Ion AmpliSeqā¢ Microbiome Health Research ā 16S rRNA Gene Pool
and the Ion AmpliSeqā¢ Microbiome Health Research ā Target Species Pool.
1
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 7
ā¢For custom assays containing only a single pool.
āThe recommended DNA sample input concentration is 0.11īng/Ī¼L for the Ion AmpliSeqā¢
Microbiome Health Research ā 16S rRNA Gene Pool.
āThe recommended DNA sample input concentration is 0.56īng/Ī¼L for the Ion AmpliSeqā¢
Microbiome Health Research ā Target Species Pool.
IMPORTANT! Ion AmpliSeqā¢ Microbiome Health Research Assay GX is only supported for use with
the GX7ā¢ Chip and Genexusā¢ Barcodes 1ā48 AS (Cat.īNo.īA54129) or Genexusā¢ Barcodes 49ā96 AS
(Cat.īNo.īA54130). The product is not supported for use with the GX5ā¢ Chip or Genexusā¢ Barcodes
1ā32īAS, 33ā64īAS, and 65ā96īAS.
Chapterī1īProduct information
Product description
1
8Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
Ion AmpliSeqā¢ Microbiome Health Research Assay GX
The Ion AmpliSeqā¢ Microbiome Health Research Assay GX (Cat.īNo.īA47022) includes the Ion
AmpliSeqā¢ Microbiome Health Research ā 16S rRNA Gene Pool and Ion AmpliSeqā¢ Microbiome
Health Research ā Target Species Pool for stool microbiome samples, and the Genexusā¢ Stripī1 and
Genexusā¢ Stripī2āAS. The contents of each kit are suīcient 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 8āstrip packs of the Genexusā¢
Stripī1 and Genexusā¢ Stripī2āAS (24 total of each strip) are provided with each kit. If needed, the
Genexusā¢ Library Strips 1 and 2āAS combo kit (Cat.īNo.īA40252) can be ordered separately.
Ion AmpliSeqā¢ Microbiome Health Research Assay GX (Cat.īNo.īA47022)
Contents Carrier color Number
of reactions Amount PartīNo. Storage
Ion AmpliSeqā¢ Microbiome Health Research Assay GX pools
Ion AmpliSeqā¢ Microbiome Health Research
ā 16S rRNA Gene Pool (position 1)
Magenta 24
(6 reactions
/tube)
4ītubes A47023 ā30Ā°C to ā10Ā°C
Ion AmpliSeqā¢ Microbiome Health Research
ā Target Species Pool (position 1)
Pale green 24
(6 reactions
/tube)
4ītubes
Genexusā¢ Library Strips 1 and 2āAS (Cat.īNo.īA40252)
Genexusā¢ Stripī1 Light red 48
(6 singleāpool
reactions/strip)
1īĆī8īstrips A46812 2Ā°C to 8Ā°C
Genexusā¢ Stripī2āAS Light blue 48
(6 singleāpool
reactions/strip)
1īĆī8īstrips A46813 ā30Ā°C to ā10Ā°C
Chapterī1īProduct information
Ion AmpliSeqā¢ Microbiome Health Research Assay GX 1
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 9
Recommended materials for use with the Ion AmpliSeqā¢
Microbiome Health Research Assay GX
Unless otherwise indicated, all materials are available through thermoļ¬sher.com. "MLS" indicates that
the material is available from ļ¬sherscientiļ¬c.com or another major laboratory supplier.
Recommended materials for nucleic acid isolation, quantiļ¬cation, and
quality control
Item Source
Nucleic acid isolation
KingFisherā¢ Puriļ¬cation System thermoļ¬sher.com/kingļ¬sher
MagMAXā¢ Microbiome Ultra Nucleic Acid Isolation Kit A42358 (tubes)
A42357 (plates)
Nucleic acid quantiļ¬cation
Qubitā¢ 4 Fluorometer[1] and one or more of the following kits:
ā¢Qubitā¢ dsDNA HS Assay Kit
ā¢Qubitā¢ 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] Qubitā¢ 2.0 Fluorometer and later are supported.
Genexusā¢ Controls
The Ion Torrentā¢ Genexusā¢ Controls kit (Cat. No. A40267) provides suīcient Genexusā¢ Control
LibraryāAS to perform four Library to Result runs. The kit also provides suīcient Genexusā¢ Control
PanelāAS and Genexusā¢ DNA Control to perform eight Nucleic Acid to Result runs.
Note: The Genexusā¢ Control LibraryāAS is barcoded with IonCodeā¢ 0101.
Component Quantity Storage
Genexusā¢ Control LibraryāAS 1 tube
ā30Ā°C to ā10Ā°CGenexusā¢ Control PanelāAS 8 carriers (white)
Genexusā¢ DNA Control 2 tubes
Chapterī1īProduct information
Recommended materials for use with the Ion AmpliSeqā¢ Microbiome Health Research Assay GX
1
10 Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
Required materials not supplied for use with the Genexusā¢
Integrated Sequencer
Genexusā¢ 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 thermoļ¬sher.com. "MLS" indicates
that the material is available from ļ¬sherscientiļ¬c.com or another major laboratory supplier. Catalog
numbers that appear as links open the web pages for those products.
Genexusā¢ Integrated Sequencer
Components Cat. No.
Genexusā¢ Integrated Sequencer A45727
General laboratory supplies and reagents
Item Source
Sorvallā¢ ST 8 Small Benchtop Centrifuge[1] (or equivalent), with
Thermo Scientiļ¬cā¢ M10 Microplate Swinging Bucket Rotor (or equivalent), and
Sealed Bucket; Capacity: 4 Standard or 2 Midi-Deepwell plates (Set ofī2) (or equivalent)
75007200
75005706
75005721
Microcentrifuge[2] MLS
MicroAmpā¢ EnduraPlateā¢ Optical 96-Well Clear Reaction Plates with Barcode 4483352
4483354
Adhesive PCR Plate Foils AB0626
2ā, 20ā, 200ā, and 1,000āĀµL pipettes and appropriate ļ¬ltered tips MLS
Nucleaseāfree 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 ā
Nuclease-free water, molecular biology grade MLS
Isopropyl alcohol, 70% solution MLS
Chapterī1īProduct information
Required materials not supplied for use with the Genexusā¢ Integrated Sequencer 1
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 11
īī(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 Genexusā¢ Strip Centrifuge Adapter.
[2] Must fit standard 0.2- and 1.5āmL microcentrifuge tubes and generate 15,000 Ć 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 2500īW output or higher.
Genexusā¢ Barcodes AS
Ion Torrentā¢ Genexusā¢ Barcodes AS are supplied in plates containing 48 dual barcodes per plate.
Item Label color Cat. No. Quantity Storage
Genexusā¢ Barcodes 1ā48 AS Blue A54129 1 plate
15Ā°C to 30Ā°C
Genexusā¢ Barcodes 49ā96 AS Blue A54130 1 plate
Note: Genexusā¢ Barcodes 1ā32īAS (Cat.īNo.īA40258), Genexusā¢ Barcodes 33ā64īAS
(Cat.īNo.īA40259), and Genexusā¢ Barcodes 65ā96īAS (Cat.īNo.īA40260) are not supported for use
with the Ion AmpliSeqā¢ Microbiome Health Research Assay GX.
Genexusā¢ Templating Strips 3āGX7ā¢ and 4
Ion Torrentā¢ Genexusā¢ Templating Strips 3āGX7ā¢ and 4 (Cat. No. A46817) are ordered as kits with
8īpairs of strips per kit.
Component Carrier color Part No. Quantity per kit Storage
Genexusā¢ Stripī3āGX7ā¢Green A46818 8 strips 2Ā°C to 8Ā°C
Genexusā¢ Stripī4 Yellow A46816 8 strips ā30Ā°C to ā10Ā°C
Chapterī1īProduct information
Required materials not supplied for use with the Genexusā¢ Integrated Sequencer
1
12 Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
Genexusā¢ Pipette Tips
Ion Torrentā¢ Genexusā¢ Pipette Tips (Cat.īNo.īA40266) 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 AmpliSeqā¢ Microbiome Health Research Assay GX, 4 racks are
suīcient for sequencing up to 4 samples and no-template controls on a single GX7ā¢ Chip lane.
Item Cat. No. Quantity Storage
Genexusā¢ Pipette Tips A40266 12 racks 15Ā°C to 30Ā°C
Genexusā¢ Sequencing Kit
The Ion Torrentā¢ Genexusā¢ Sequencing Kit (Cat. No. A40271) provides reagents and solutions suīcient
to sequence up to 2īfull chips.
Component Part No. Quantity Storage
Genexusā¢ Cartridge A40272 2 cartridges ā30Ā°C to ā10Ā°C
Genexusā¢ Bottle 2 A40273 4ībottles 15Ā°C to 30Ā°C
Genexusā¢ Bottles 1 and 3 A40274 2ībottles each
(4 bottles total)
Chapterī1īProduct information
Required materials not supplied for use with the Genexusā¢ Integrated Sequencer 1
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 13
GX7ā¢ Chip and Genexusā¢ Coupler
The GX7ā¢ Chip and Genexusā¢ Coupler (Cat. No. A46731) are ordered as a set that contains 2 chips
and 2 couplers, suīcient for up to 8 sequencing runs.
Component Part No. Quantity Storage
GX7ā¢ Chip
7
100093568 2 chips 15Ā°C to 30Ā°C
Genexusā¢ Coupler 100081252 2 couplers
Genexusā¢ Conical Bottles
Genexusā¢ 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 Genexusā¢ Integrated Sequencer User Guide (Pub. No. MAN0017910). .
Component Quantity Storage
Genexusā¢ Conical Bottles 5 bottles 15Ā°C to 30Ā°C
Chapterī1īProduct information
Required materials not supplied for use with the Genexusā¢ Integrated Sequencer
1
14 Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
Nucleic Acid to Result workļ¬ow
The Nucleic Acid to Result workļ¬ow starts from puriļ¬ed and quantiļ¬ed nucleic acid samples. Puriļ¬ed
nucleic acid samples are pipet into a 96-well sample input plate and then loaded into the Genexusā¢
Integrated Sequencer for library preparation, templating, and sequencing.
Nucleic Acid to Result
Prepare puriļ¬ed DNA
We recommend the MagMAXā¢ Microbiome Ultra Nucleic Acid Isolation Kit for
extraction of DNA from stool samples for use in Nucleic Acid to Result runs.
Use a Qubitā¢ Fluorometer to accurately measure sample concentration.
Note: A KingFisherā¢ puriļ¬cation system can be used to automate extraction of
DNA from stool samples.
up to
3īhr
Enter samples (pageī24)
Before you plan a run in Genexusā¢ 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 (pageī25)
In the Genexusā¢ Software, plan a Nucleic Acid to Result run to create a
sequencing run that starts with nucleic acid samples. The run plan deļ¬nes the
samples, assays, sequencing reagents, and the number of sequencing chip
lanes that are needed.
5 min
Prepare sample input plate (pageī28)
Transfer puriļ¬ed DNA to a 96-well sample input plate for loading into the
Genexusā¢ Integrated Sequencer. Add positive and negative controls to the
sample input plate if included in the run plan.
10 min
Sequence the puriļ¬ed nucleic acid sample on the
Genexusā¢ Integrated Sequencer (pageī35)
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
24īhr
Chapterī1īProduct information
Nucleic Acid to Result workļ¬ow 1
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 15
Before you begin
ā Proceduralīguidelines ................................................................. 16
ā Guidelines for nucleic acid isolation, quantiļ¬cation, quality control, and inputīamount ......... 17
ā Guidelines for Genexusā¢ Integrated Sequencer operation ................................. 17
ā Guidelines for panel and reagent use andīhandling ....................................... 18
ā Guidelines for preventingīcontamination ................................................. 19
ā Before each use of theīkit ............................................................. 20
ā Precautions .......................................................................... 21
ā Install software and assay deļ¬nition ļ¬le updates .......................................... 21
Procedural guidelines
ā¢Use only the reagents and supplies that have been validated for the Ion AmpliSeqā¢ Microbiome
Health Research Assay GX. For a list of validated reagents and supplies, see āRequired materials
not supplied for use with the Genexusā¢ Integrated Sequencerā on pageī11 and āRecommended
materials for use with the Ion AmpliSeqā¢ Microbiome Health Research Assay GXā on pageī10.
ā¢Use good laboratory practices to minimize cross-contamination of products. Keep all tubes sealed
until immediately before loading onto the Genexusā¢ Integrated Sequencer.
ā¢Use controls to identify or rule out reagent contamination. For a list of validated controls, see
āGenexusā¢ Controlsā on pageī10.
ā¢Each GX7ā¢ 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.
ā¢Minimize freezeāthaw cycles of the Ion AmpliSeqā¢ Microbiome Health Research ā 16S rRNA Gene
Pool, Ion AmpliSeqā¢ Microbiome Health Research ā Target Species Pool tubes. Thaw only the
number of panel tubes that are required for a given experiment and keep the thawed panels at 4Ā°C
until ready to use. Store unused panels at ā30Ā°C to ā10Ā°C.
Note: One set of panels (Ion AmpliSeqā¢ Microbiome Health Research ā 16S rRNA Gene Pool, Ion
AmpliSeqā¢ Microbiome Health Research ā Target Species Pool tubes) is suīcient for processing
up to 4īsamples.
ā¢Do NOT store the Ion AmpliSeqā¢ Microbiome Health Research ā 16S rRNA Gene Pool, Ion
AmpliSeqā¢ Microbiome Health Research ā Target Species Pool tubes, and the Genexusā¢ Stripī1
and Genexusā¢ Stripī2āAS on the Genexusā¢ Integrated Sequencer for more than 24 hours before
starting an instrument run.
2
16 Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
Guidelines for nucleic acid isolation, quantiļ¬cation, quality
control, and input amount
These are general guidelines for manual isolation and quantiļ¬cation 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.
ā¢For validated kits to use for isolation, quantiļ¬cation, and quality control of nucleic acid samples,
see āRecommended materials for nucleic acid isolation, quantiļ¬cation, and quality controlā on
pageī10.
ā¢Quantiļ¬cation methods such as densitometry (for example, using a NanoDropā¢ spectrophotometer)
are not recommended, because they are not speciļ¬c for DNA. Use of these methods can lead
to gross overestimation of the concentration of sample nucleic acid, under-seeding of the target
ampliļ¬cation reaction, and low library yields.
ā¢The recommended DNA sample input concentration is 0.11īng/Ī¼L for the Ion AmpliSeqā¢
Microbiome Health Research ā 16S rRNA Gene Pool and 0.56īng/Ī¼L for the Ion AmpliSeqā¢
Microbiome Health Research ā Target Species Pool.
Guidelines for Genexusā¢ Integrated Sequencer operation
ā¢Follow guidance that is provided by Genexusā¢ Software when you plan a run to determine which
consumables must be loaded and which consumables can be reused from a previous run.
ā¢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
ampliļ¬cation reactions per run
One library strip pair has the reagents necessary for 6 target
ampliļ¬cation reactions. With a maximum of 8 library strip pairs
loaded, a maximum of 24īsamples can be run using the Ion
AmpliSeqā¢ Microbiome Health Research Assay GX, which contains
2īprimer 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 GX7ā¢ Chip.
The minimum read count per
sample for an assay
The minimum read count per sample parameter is set during assay
creation.
ā¢One library strip pair is needed for each primer tube position 1ā8 that is ļ¬lled in a run.
ā¢One template strip pair is needed for each chip lane that is used in a run.
Chapterī2īBefore you begin
Guidelines for nucleic acid isolation, quantiļ¬cation, quality control, and input amount 2
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 17
ā¢Consumables are conļ¬gured to support sample batch sizes in multiples of 4-6 samples. The most
eīcient use of consumables occurs when samples are run in multiples of four or six.
ā¢Each GX7ā¢ Chip can accommodate up to 48 microbiome DNA samples for analysis, 24 samples if
running both pools, across 2īlanes per run.
ā¢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.
ā¢You can remove a chip in one of the following situations.
āAfter 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.
āWhen 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.
ā¢The Genexusā¢ Integrated Sequencer can track used and unused barcodes in barcode plates in
Genexusā¢ 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 suīcient number of barcodes are available on
the plate.
ā¢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
ā¢Use only the reagents and supplies that have been recommended in āGeneral laboratory supplies
and reagentsā on pageī11 and āRecommended materials for nucleic acid isolation, quantiļ¬cation,
and quality controlā on pageī10.
ā¢Keep panel tubes capped until immediately before loading in the Genexusā¢ Integrated Sequencer.
ā¢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.
ā¢Equilibrate Genexusā¢ Stripī1 and Genexusā¢ Stripī3āGX5ā¢ (or Genexusā¢ Stripī3āGX7ā¢) at room
temperature for 30 minutes before loading in the sequencer.
ā¢Thaw Genexusā¢ Stripī2āAS (or Genexusā¢ Stripī2āHD) and Genexusā¢ Stripī4 on ice for 30 minutes.
IMPORTANT! Ensure that strip contents are completely thawed before loading in the sequencer.
ā¢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 Genexusā¢ Strip Centrifuge Adapter to hold strips during centrifugation.
For information on obtaining and using the Genexusā¢ Strip Centrifuge Adapter, see āCentrifuge
library and templating reagent strips using the Genexusā¢ Strip Centrifuge Adapterā on pageī34.
Chapterī2īBefore you begin
Guidelines for panel and reagent use and handling
2
18 Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
ā¢Keep thawed panel, Genexusā¢ Stripī2āAS (or Genexusā¢ Stripī2āHD), Genexusā¢ Stripī4, and the
sample plate on ice or at 4Ā°C until ready to load in the sequencer.
ā¢Do not freezeāthaw 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 ā30Ā°C to
ā10Ā°C.
ā¢If you are using an assay that uses Ion AmpliSeqā¢ 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 onāinstrument library preparation workļ¬ow.
ā¢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
ā¢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.
ā¢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.
ā¢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
ā¢If you are preparing samples and sample plates in a hood (recommended), illuminate the hood with
UV light for 15 minutes before use.
ā¢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 DNAZapā¢ decontamination solutions. Wipe with solutionī1 ļ¬rst, 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.
ā¢Before and after a run, sequentially wipe instrument deck surfaces with lint-free wipes moistened
with the two DNAZapā¢ 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.
ā¢We recommend that you centrifuge the sample plate after sealing. Ensure that the centrifuge has
been wiped down and cleaned before centrifuging the plate.
Chapterī2īBefore you begin
Guidelines for preventing contamination 2
Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide 19
Workļ¬ow tips
ā¢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.
ā¢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.
ā¢If possible, have a pipettor and tips reserved for dispensing only NTC.
ā¢Before disposal, close or cap used sample and positive control tubes to avoid creation of aerosols.
ā¢Avoid touching the foil seals of the reagent strips, barcode plate, and sample plate.
ā¢When loading the sequencer deck for a run, install the sample plate last.
ā¢After a run, seal the PCR ampliļ¬cation plate with a foil seal before removing the plate from the PCR
ampliļ¬cation 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:
ā¢Thaw Ion AmpliSeqā¢ Microbiome Health Research ā 16S rRNA Gene Pool, Ion AmpliSeqā¢
Microbiome Health Research ā Target Species Pool on ice or at 4Ā°C for at least 30 minutes and
keep on ice until immediately before loading onto the Genexusā¢ Integrated Sequencer.
ā¢Equilibrate Genexusā¢ Stripī1 and Genexusā¢ Stripī3āGX7ā¢ at room temperature for at least
30 minutes before loading onto the Genexusā¢ Integrated Sequencer.
ā¢Thaw Genexusā¢ Stripī2āAS and Genexusā¢ Stripī4 on ice or at 4Ā°C for at least 30 minutes and keep
on ice until immediately before loading onto the Genexusā¢ 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 āBefore you beginā on pageī31.
Note: If tapping fails to dislodge a bubble, you can dislodge large bubbles using the technique that
is described in āCentrifuge library and templating reagent strips using the Genexusā¢ Strip Centrifuge
Adapterā on pageī34.
Chapterī2īBefore you begin
Before each use of the kit
2
20 Ion AmpliSeqā¢ Microbiome Health Research Assay GX User Guide
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