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

Brand
Thermo Fisher Scientific
Model
Ion AmpliSeq Microbiome Health Research Assay GX
Type
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.
©2023 Thermo Fisher Scientific Inc. All rights reserved.
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
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
Page is loading ...
  • Page 1 1
  • Page 2 2
  • Page 3 3
  • Page 4 4
  • Page 5 5
  • Page 6 6
  • Page 7 7
  • Page 8 8
  • Page 9 9
  • Page 10 10
  • Page 11 11
  • Page 12 12
  • Page 13 13
  • Page 14 14
  • Page 15 15
  • Page 16 16
  • Page 17 17
  • Page 18 18
  • Page 19 19
  • Page 20 20
  • Page 21 21
  • Page 22 22
  • Page 23 23
  • Page 24 24
  • Page 25 25
  • Page 26 26
  • Page 27 27
  • Page 28 28
  • Page 29 29
  • Page 30 30
  • Page 31 31
  • Page 32 32
  • Page 33 33
  • Page 34 34
  • Page 35 35
  • Page 36 36
  • Page 37 37
  • Page 38 38
  • Page 39 39
  • Page 40 40
  • Page 41 41
  • Page 42 42
  • Page 43 43
  • Page 44 44
  • Page 45 45
  • Page 46 46
  • Page 47 47
  • Page 48 48
  • Page 49 49
  • Page 50 50
  • Page 51 51
  • Page 52 52
  • Page 53 53
  • Page 54 54
  • Page 55 55
  • Page 56 56
  • Page 57 57
  • Page 58 58
  • Page 59 59
  • Page 60 60
  • Page 61 61
  • Page 62 62
  • Page 63 63
  • Page 64 64
  • Page 65 65
  • Page 66 66
  • Page 67 67
  • Page 68 68
  • Page 69 69
  • Page 70 70
  • Page 71 71
  • Page 72 72
  • Page 73 73
  • Page 74 74
  • Page 75 75
  • Page 76 76
  • Page 77 77
  • Page 78 78
  • Page 79 79
  • Page 80 80
  • Page 81 81
  • Page 82 82
  • Page 83 83
  • Page 84 84
  • Page 85 85
  • Page 86 86
  • Page 87 87
  • Page 88 88

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

Brand
Thermo Fisher Scientific
Model
Ion AmpliSeq Microbiome Health Research Assay GX
Type
User guide
Download PDF
report

Ask a question and I''ll find the answer in the document

Finding information in a document is now easier with AI

Related papers

Other documents