AgriSeq HTS Library Kit MAN0015971 Revision€¦ · Only 12, 24, 36, 48, 60, 72 or 96 samples can...

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

AgriSeq™ HTS Library KitUSER GUIDE

For high-throughput AgriSeq™ library preparation from plant andanimal samples

for use with:IonCode™ Barcode AdaptersBovine ISAG SNP Parentage PanelIon 540™ Chip KitIon S5™ SystemIon S5™ XL SystemIon Chef™ System

Catalog Numbers A34143, A34144Publication Number MAN0015971

Revision A.0

Manufacturer: Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008

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

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOURUSE OF IT.

Revision history: Pub. No. MAN0015971

Revision Date DescriptionA.0 13 July 2017 New document

Limited Use Label License No. 666: AgriSeq Products: Notice to Purchaser: The purchase of this product conveys to the purchaser the limited, non-transferable right for the purchaser to use the product only to perform for the sole benefit of the purchaser internal agricultural research conductedwith plants, agricultural animals or companion animals only. No right to resell this product or any of its components is conveyed expressly, byimplication, or by estoppel. This product is not for use with human samples and/or in commercial applications of any kind, including, withoutlimitation, quality control and commercial services such as reporting the results of purchaser's activities for a fee or any other form of consideration.For information on obtaining additional rights, please contact [email protected] or Licensing and Commercial Supply, Thermo FisherScientific, 5823 Newton Drive, Carlsbad, CA, 92008, United States.Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Agencourt and AMPure aretrademarks of Beckman Coulter, Inc.

©2017 Thermo Fisher Scientific Inc. All rights reserved.

mailto: [email protected]

Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ CHAPTER 2 96–well plate format procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Normalize the input DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Prepare the DNA target amplification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Prepare amplicons for barcode addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Add barcodes to the sample libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Pool the libraries: 96‑well plate format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Prepare Normalization Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Purify the unamplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Normalize the libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Add Normalization Reagent to the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Add Normalization Beads and wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Elute the normalized library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Final library pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ CHAPTER 3 384–well plate format procedure . . . . . . . . . . . . . . . . . . . . . . . 21

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Normalize the input DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Prepare the DNA target amplification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Prepare amplicons for barcode addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

AgriSeq™ HTS Library Kit User guide 3

Add barcodes to the sample libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Pool the libraries: 384‑well plate format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Prepare Normalization Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Purify the unamplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Normalize the libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Add Normalization Reagent to the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Add Normalization Beads and wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Elute the normalized library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Final library pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Library yield and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Low amplicon uniformity (DNA only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

■ APPENDIX B Ion Chip™ capacities for AgriSeq™ DNA librariesrun on the Ion 540™ Chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Contents

4 AgriSeq™ HTS Library Kit User guide

Product information

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

Product description

The Applied Biosystems™ AgriSeq™ HTS Library Kit contains reagents for generatingsequencing libraries for the genotyping of plant and animal samples.

This user guide describes how to prepare libraries using the AgriSeq™ HTS LibraryKit in a 96‑ or 384‑well format.

The AgriSeq™ HTS Library Kit requires Ion AmpliSeq™ panels that are specificallydesigned to work with the AgriSeq™ chemistry. Ion AmpliSeq™ panels provide poolsof primers for the amplification of target sequences ranging from 100 to 5000amplicons. The primers contain proprietary modifications that are required forefficient sequencing with the AgriSeq™ chemistry.

Contents and storage

ContentsA34144 A34143

Storage960 reactions 9600 reactions

AgriSeq™ HTS Library Reagents[1]

AgriSeq™ Amplification Mix 2.3 mL 22 mL –30°C to – 10°C

Pre-ligation Enzyme 1300 µL 13.5 mL

Barcoding Buffer 2.3 mL 22.5 mL

Barcoding Enzyme 1500 µL 15 mL

Normalization Master Mix 6.75 mL 5 x 13.75 mL

Low TE 4.5 mL 46 mL –30°C to – 10°C[2]

Nuclease-free Water 1300 µL 13.5 mL

AgriSeq™ Normalization Reagents[3]

Normalization Primers 200 µL 3 mL 2—8°C

Normalization Beads 550 µL 6.5 mL

Normalization Reagent 1700 µL 17.5 mL

1


ContentsA34144 A34143

Storage960 reactions 9600 reactions

Normalization Wash 2—8°C36.5 mL 120 mL

Normalization Elution Solution 7.5 mL 79 mL

[1] Shipped at –20°C. Store as directed.[2] You can also store the Low TE and Nuclease-free water at 4°C or room temperature.[3] Shipped at room temperature. Store as directed.

Note: The reagents include overage for use in automation protocols. ContactTechnical Support for details (see “Customer and technical support“ on page 41 forcontact information).

Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Item Source

IonCode™ Barcode Adapters 1–384 Kit A31173

Ion AmpliSeq™ Panels (select one of the following):

Bovine ISAG SNP Parentage Panel A35297

Custom panels: AgriSeq™ Bioinformatic design service Contact your salesrepresentative.[1]

Other materials

DynaMag™–96 Side Magnet 12331D

MicroAmp™ EnduraPlate™ Optical 96-Well Clear ReactionPlates with Barcode

4483354

For 384-sample procedure only: MicroAmp™ EnduraPlate™

Optical 384-Well Clear Reaction Plates with Barcode4483285

One of the following (or equivalent):

• Veriti™ 96-well Thermal Cycler

• GeneAmp™ PCR System 9700[2]

4375786

Plate centrifuge MLS

MicroAmp™ Adhesive Film 4306311

MicroAmp™ Optical Film Compression Pad 4312639

Agencourt™ AMPure™ XP Kit Beckman Coulter™

A63880

A63881

Chapter 1 Product informationRequired materials not supplied1


http://www.thermofisher.com

http://fisherscientific.com

Item Source

Absolute ethanol MLS

(Optional) Recommended for DNA isolation[3]

PureLink™ gDNA purification kit for your sample type Various[3]

MagMAX™ Plant DNA Isolation Kit A32549

DNA Extract All Reagents Kit 4403319

MagMAX™ CORE Nucleic Acid Purification Kit A32700

Recommended for DNA quantification

Qubit™ dsDNA HS Assay Kit

or

Quant-iT™ DNA Assay Kit, High Sensitivity

Q32851

or

Q33120

[1] Panels can also be ordered on www.ampliseq.com.[2] The GeneAmp™ PCR System 9700 has been discontinued, but is still supported. [3] To select a kit, see https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/

dna-and-rna-selection-guide.html.

Chapter 1 Product informationRequired materials not supplied 1


https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-and-rna-selection-guide.html

https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-and-rna-selection-guide.html

Workflow

AB

CD

EF

GH

21

34

56

7 8 9 10 11 12

Amplify targets to generate AgriSeq™ libraries (2 hours)

q

AB

CD

EF

GH

21

34

56

7 8 9 10 11 12

Prepare libraries for barcoding (40 minutes)

q

AB

CD

EF

GH

21

34

56

7 8 9 10 11 12

Barcode libraries (40 minutes)

q

Pool libraries (10 minutes)

q

Purify the libraries (1 hour)

q

Normalize the purified libraries (30 minutes)

Chapter 1 Product informationWorkflow1


96–well plate format procedure

■ Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

■ Normalize the input DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

■ Prepare the DNA target amplification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

■ Prepare amplicons for barcode addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

■ Add barcodes to the sample libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ Pool the libraries: 96‑well plate format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ Prepare Normalization Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

■ Purify the unamplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

■ Normalize the libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■ Final library pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Note: The term 96‑well format applies to samples prepared using a 96‑well formatbefore pooling. After pooling, only 8 wells of the 96‑well plate with the pooledsamples are used.

IMPORTANT! If you are running fewer than 96 samples, you must run full rows.Only 12, 24, 36, 48, 60, 72 or 96 samples can be run.

Procedural guidelines

• Store aliquots to minimize freeze‑thawing of the AmpliSeq™ Panels. Panels can bestored at 4°C for one year.

• Use good laboratory practices to minimize cross‑contamination of products. Ifpossible, perform PCR setup in an area or room that is separate from templatepreparation. Always change pipette tips between samples.

• Use a calibrated thermal cycler.• Pipet viscous solutions slowly and ensure complete mixing by vigorous vortexing

or pipetting up and down several times.• To prevent cross‑contamination and aerosolization, before removing plate seals,briefly centrifuge the plate to collect contents.

2


Before each use of the kit

• Thaw components that contain enzymes (such as the AgriSeq™ AmplificationMix, Pre‑ligation Enzyme, Barcoding Enzyme, and Normalization Master Mix) onice and keep on ice during the procedure. All other components, includingprimer pools, can be thawed at room temperature. Gently vortex and centrifugebefore use.

• If there is visible precipitate in the Barcoding Buffer after thawing, vortex or pipetup and down at room temperature to dissolve.

Normalize the input DNA

IMPORTANT! All DNA inputs must be normalized to 10 ng per reaction. Failure tonormalize DNA input can negatively affect call rates.

Use high‑quality gDNA. See “Required materials not supplied“ on page 6 forrecommended isolation kits.

1. Quantify the input DNA.• We recommend the Qubit™ dsDNA HS Assay Kit (Cat. No. Q32851 or

Q32854) or the Quant‑iT™ DNA Assay Kit, High Sensitivity (Cat. No.Q33120) for quantification of DNA.

• Do not use quantification methods such as densitometry (for example, usinga NanoDrop™ spectrophotometer), because they are not specific for DNA.Use of these methods can lead to gross overestimation of the concentrationof sample DNA, under‑seeding of the target amplification reaction, and lowlibrary yields.

2. Normalize each sample of DNA to a final concentration of 3.3 ng/µL (3 µL).

Note: If the DNA concentration is higher than 3.3 ng/µL, dilute the DNA to afinal concentration of 3.3 ng/µL with nuclease‑free water.

Chapter 2 96–well plate format procedureBefore each use of the kit2


Prepare the DNA target amplification reaction

1. Prepare the Amplification Reaction Mix in a microcentrifuge tube.

IMPORTANT! The AgriSeq™ Amplification Mix is viscous. Pipet slowly and mixthoroughly.

Component Volume per well(2X primer pool)

Volume per well(5X primer pool)

AgriSeq™ Amplification Mix(red cap) 2 µL 2 µL

2X AmpliSeq™ Primer Pool 5 µL —

5X AmpliSeq™ Primer Pool — 2 µL

Nuclease-free water — 3 µL

Total Amplification Reaction Mix perwell

7 µL 7 µL

Note:· Scale up the volumes for the number of reactions being run.· Include 10% overage.

2. Add 7 µL of the Amplification Reaction Mix to each well of a MicroAmp™

EnduraPlate™ Optical 96–Well Clear Reaction Plate (reaction plate).

Note: The reaction plate does not contain IonCode™ Barcode Adapters, whichare added later in the procedure. The barcode in the product name refers to thebarcode for the plate.

3. Add 3 µL of normalized DNA to each well containing Amplification ReactionMix. (See “Normalize the input DNA“ on page 10.)The total volume for each well is 10 µL.

4. Seal the plate with a MicroAmp™ Clear Adhesive Film, vortex thoroughly, thenbriefly centrifuge to collect contents.

Not a stopping point. Proceed immediately to the next section.

Chapter 2 96–well plate format procedurePrepare the DNA target amplification reaction 2


Amplify the targets

1. Place a MicroAmp™ Optical Film Compression Pad on the reaction plate, thenload the plate in a thermal cycler.

2. Run the following program (10‑µL reaction size).

Stage Step Temperature Time

Hold Activate theenzyme

99°C 2 minutes

Cycle; set numberaccording to thefollowing tables

Denature 99°C 15 seconds

Anneal and extend 60°C 4 or 8 minutes[1]

Hold — 10°C Hold

[1] Use 4 minutes for £1536 primer pairs per pool; 8 minutes for 1,537–5,000 primer pairs per pool.

IMPORTANT! We recommend selecting the number of primer pairs per pool toensure that the mean coverage depth is greater than 100X. There can besignificant marker drop-off when the mean coverage depth is less than 100X. Fordetails, see Appendix B, “Ion Chip™ capacities for AgriSeq™ DNA libraries runon the Ion 540™ Chip“.

Primer pairs per pool (see notes below) Recommended number of amplificationcycles

97–192 18

193–384 17

385–768 16

769–1,536 15

1,537–3,072 14

3,073–5,000 13

STOPPING POINT Target amplification reactions can be stored at 10°C overnighton the thermal cycler. For longer term, store at −20°C.

Chapter 2 96–well plate format procedureAmplify the targets2


Prepare amplicons for barcode addition

1. Prepare the Pre‑ligation Enzyme Reaction Mix in a microcentrifuge tube, thenmix well by pipetting.

Component Volume per well

Pre-ligation Enzyme 1 µL

Nuclease-free Water 1 µL


2. Briefly centrifuge the reaction plate to collect contents. The reaction plate wasprepared in the previous section, "Amplify the targets".

3. Carefully remove the plate seal, then add 2 µL of Pre‑ligation Enzyme ReactionMix (from step 1) to each amplified sample. The total volume per well is 12 µL(10 µL of sample + 2 µL of Pre‑ligation Enzyme Reaction Mix).

4. Seal the reaction plate with a MicroAmp™ Adhesive Film, vortex thoroughly,then briefly centrifuge to collect contents.

5. Place a MicroAmp™ Optical Film Compression Pad on the reaction plate, thenload in a thermal cycler.

6. Run the following program (12–µL reaction volume):

Temperature Time

50°C 10 minutes[1]

55°C 10 minutes [1]

60°C 20 minutes

10°C Hold (for up to 1 hour)

[1] Increase to 20 minutes for panels over 1,536 primer pairs/pool.


Chapter 2 96–well plate format procedurePrepare amplicons for barcode addition 2


Add barcodes to the sample libraries

If there is visible precipitate in the Barcoding Buffer or the tube cap after thawing,vortex or pipet up and down at room temperature to resuspend before pipetting.

1. Prepare the Barcode Reaction Mix in a microcentrifuge tube:

Components Volume per well

Barcoding Buffer 2 µL

Barcoding Enzyme 1 µL

Total volume per well 3 µL


2. Briefly centrifuge the reaction plate to collect the contents.

3. Carefully remove the plate seal, then discard.

4. Prepare the IonCode™ Barcode Adapters.a. Briefly centrifuge the IonCode™ Barcode Adapters plate to collect contents.

b. Carefully remove, then discard the plate seal.

5. Add 1 µL of an IonCode™ Barcode Adapter to each sample well of the reactionplate, using a different barcode adapter for each sample.

6. Seal the IonCode™ Barcode Adapters plate with a new MicroAmp™ AdhesiveFilm, then store as recommended.

7. Add 3 µL of the Barcode Reaction Mix to each sample well of the reaction plate.




Temperature Time

22°C 30 minutes

72°C 10 minutes

10°C Hold[1]

[1] Up to 24 hours

STOPPING POINT Libraries can be stored at –20°C.

Chapter 2 96–well plate format procedureAdd barcodes to the sample libraries2


Pool the libraries: 96‑well plate format

IMPORTANT! Pooling can alter coverage depth. There can be significant markerdrop-off when the mean coverage depth is less than 100X. For details, see Appendix B,“Ion Chip™ capacities for AgriSeq™ DNA libraries run on the Ion 540™ Chip“.

1. Briefly centrifuge the plate to collect the contents.

2. Combine 5 µL from each well of a single row of the 96‑well reaction plate (12samples) into a single well of an 8‑strip tube, then mix well.

3. Transfer 45 µL from each well of the 8‑strip tube to a new well on a 96‑well PCRplate (sample plate).

Note:· Twelve 96–well plates can be loaded and cleaned on the same post‑pooling

plate.· If running fewer than 96 samples, adjust the number of pools accordingly. For

example, if you are running 60 samples, only 5 pools are created.

Chapter 2 96–well plate format procedurePool the libraries: 96‑well plate format 2


Prepare Normalization Reaction Mix

Prepare Normalization Reaction Mix in a microcentrifuge tube before AMPure™

library clean‑up, so that the normalization procedure can be started immediately afterpurification of the unamplified libraries, which avoids over‑drying the beads at theend of the purification.

Component Volume per reaction (96-well format)

Normalization Master Mix 75 µL

Normalization Primers 3 µL


Store on ice (or at 4°C) until use.

Purify the unamplified libraries

IMPORTANT!· Bring the AMPure™ XP reagent to room temperature and vortex to disperse the

beads before use. The AMPure™ XP Reagent is purchased separately (see “Requiredmaterials not supplied“ on page 6).

· Pipet the solution slowly.· Use freshly prepared 70% ethanol for the next steps. (Combine 315 µL of 100%

Ethanol with 135 µL of nuclease‑free water per sample.)

1. To each well of the sample plate with the pooled libraries (prepared in step 3, “Pool the libraries: 96‑well plate format“ on page 15), add 67.5 µL of theAgencourt™ AMPure™ XP Reagent, then pipette up and down 5 times tothoroughly mix the bead suspension with the DNA.

Note: Only the wells containing the pooled libraries should be processed.

2. Incubate the mixture for 5 minutes at room temperature.

3. Place the plate on a magnetic stand such as the DynaMag™–96 Side Magnet, thenincubate for 5 minutes or until solution clears.

4. Carefully remove, then discard the supernatant without disturbing the pellet.

5. Add 112 µL of freshly prepared 70% ethanol to each well of the sample platewith pooled libraries, move the plate side‑to‑side in the two positions of themagnet to wash the beads, then remove and discard the supernatant. Do notdisturb the pellet.

Note: If your magnet does not have two positions for shifting the beads, removethe plate from the magnet and gently pipet up and down five times (with thepipettor set to at least half the total volume), then return the plate to the magnetand incubate for 2 minutes or until the solution clears.

Chapter 2 96–well plate format procedurePrepare Normalization Reaction Mix2


6. Repeat step 5 for a second wash.

IMPORTANT! Ensure that all ethanol droplets are removed from the wells.

7. Remove the plate from the magnetic stand, then add 45 µL of Low TE to eachwell of the sample plate with pooled libraries.

8. Pipette up and down 5 times to thoroughly mix.

9. Incubate at room temperature for 2 minutes.

10. Add 54 µL of the Agencourt™ AMPure™ XP Reagent to each well of the sampleplate with pooled libraries, then pipet up and down 5 times to thoroughly mixthe bead suspension with the DNA.








16. Dry the beads, uncovered, at 40°C for 1 minute. Do not over-dry.

IMPORTANT! Proceed immediately to Normalization. Do not allow the beads tosit uncovered and over‑dry.

Normalize the libraries

• Warm all the AgriSeq™ Normalization Reagents to room temperature.• Vortex and centrifuge all reagents before use.

Before you begin

Chapter 2 96–well plate format procedureNormalize the libraries 2


1. Add 78 µL of Normalization Reaction Mix to each bead pellet. TheNormalization Reaction Mix was prepared in “Prepare Normalization ReactionMix“ on page 16.

2. Seal the plate with a MicroAmp™ Adhesive Film, vortex thoroughly, thencentrifuge to collect contents. Alternatively, mix by pipetting at least half the totalvolume up and down at least 5 times before sealing the plate.

3. Place a MicroAmp™ Optical Film Compression Pad on the plate, then load in thethermal cycler. Run the following program (78‑µL reaction volume).

Note: Library amplification takes place in the presence of the AMPure® XPbeads.


Hold Activate enzyme 98°C 2 minutes

9 cycles Denature 98°C 15 seconds

Anneal/Extend 64°C 1 minute

Hold — 10°C Hold (up to 1hour)

1. After thermal cycling, briefly centrifuge the plate to collect the liquid in the wells.

2. Remove the seal from the plate, then add 15 µL of Normalization Reagent to eachlibrary amplification reaction.

3. Mix by pipetting at least half of the total volume up and down at least 5 times.


1. If not already prepared, wash the Normalization Beads.a. Thoroughly mix the Normalization Beads, transfer 4.5 µL/reaction to a fresh

1.5–mL microcentrifuge tube, then capture the beads on a magnet for3 minutes.

b. Remove, then discard the supernatant.

c. Add Normalization Wash Buffer to each pellet (9 µL/reaction).

Prepared beads can be stored at 4°C for up to 6 months.

2. Mix the pre‑washed Normalization Beads by gentle vortexing or pipetting upand down.

3. Add 9 µL of washed beads to each well containing the normalization reaction.

4. Mix by pipetting at least half the total volume up and down at least 5 times.


6. Place the plate in the magnet, then incubate for 2 minutes or until the solution isclear.

Amplify the library

Add NormalizationReagent to theamplified library

Add NormalizationBeads and wash

Chapter 2 96–well plate format procedureNormalize the libraries2



8. Add 112 µL of Normalization Wash Buffer to each well containing thenormalization reaction.

9. Place the plate on a magnet, then move the plate side‑to‑side in the two positionsof the magnet to wash the beads.


10. Incubate the plate on the magnet for 2 minutes or until the solution is clear.

11. With the plate still in the magnet, carefully remove, then discard the supernatantwithout disturbing the pellet.

12. Repeat the bead wash as described in step 8–step 11.

IMPORTANT! Ensure that as much wash buffer as possible is removed withoutdisturbing the pellet.

1. Remove the plate from the magnet, then add 75 µL of Normalization ElutionBuffer to each pellet.

2. Seal the plate with MicroAmp™ Clear Adhesive Film, vortex thoroughly, thencentrifuge to collect contents. Alternatively, mix by pipetting at least half the totalvolume up and down at least 5 times before sealing the plate.

Note: Centrifuge with sufficient force to collect droplets, but not to pellet thebeads. If beads are pelleted, vortex again and centrifuge more gently.

3. Elute the library by incubating in a thermal cycler at 32°C for 5 minutes.

4. Place the plate in the magnet, then incubate at room temperature for 5 minutes oruntil the solution is clear.

5. (Optional) To store unpooled libraries, transfer the supernatant to a new well forstorage.

STOPPING POINT

· The supernatant can be stored at 4–8°C for 1 month.· For longer term storage, store libraries at −20°C.

Elute thenormalized library



Final library pooling

Combine 50 µL of each normalized library in a single pooled sample of 400 µL.

Proceed to template preparation. For details, see the user guide for your templatepreparation kit.

STOPPING POINT

· The supernatant contains the Normalized library at ~200 pM.· Store the remainder of the libraries at 4–8°C for 1 month.· For longer term storage, store libraries at −20°C.

Chapter 2 96–well plate format procedureFinal library pooling2


384–well plate format procedure

■ Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

■ Before each use of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ Normalize the input DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ Prepare the DNA target amplification reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■ Amplify the targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

■ Prepare amplicons for barcode addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

■ Add barcodes to the sample libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

■ Pool the libraries: 384‑well plate format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

■ Prepare Normalization Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■ Purify the unamplified libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■ Normalize the libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

■ Final library pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Note: The term 384‑well format applies to samples prepared using a 384‑well formatbefore pooling. After pooling, only 24 wells of the 96‑well plate with the pooledsamples are used.

IMPORTANT! If you are running fewer than 384 samples, you must run full rows.Only 16, 32, 48, 64, 80, 96, 108, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304,320, 336, 352, 368, or 384 samples can be run.

Procedural guidelines

• Store aliquots to minimize freeze‑thawing of the AmpliSeq™ Panels. Panels can bestored at 4°C for one year.

• Use good laboratory practices to minimize cross‑contamination of products. Ifpossible, perform PCR setup in an area or room that is separate from templatepreparation. Always change pipette tips between samples.

• Use a calibrated thermal cycler.• Pipet viscous solutions slowly and ensure complete mixing by vigorous vortexing

or pipetting up and down several times.• To prevent cross‑contamination and aerosolization, before removing plate seals,briefly centrifuge the plate to collect contents.

3


Before each use of the kit

• Thaw components that contain enzymes (such as the AgriSeq™ AmplificationMix, Pre‑ligation Enzyme, Barcoding Enzyme, and Normalization Master Mix) onice and keep on ice during the procedure. All other components, includingprimer pools, can be thawed at room temperature. Gently vortex and centrifugebefore use.

• If there is visible precipitate in the Barcoding Buffer after thawing, vortex or pipetup and down at room temperature to dissolve.

Normalize the input DNA

IMPORTANT! All DNA inputs must be normalized to 10 ng per reaction. Failure tonormalize DNA input can negatively affect call rates.

Use high‑quality gDNA. See “Required materials not supplied“ on page 6 forrecommended isolation kits.

1. Quantify the input DNA.• We recommend the Qubit™ dsDNA HS Assay Kit (Cat. No. Q32851 or

Q32854) or the Quant‑iT™ DNA Assay Kit, High Sensitivity (Cat. No.Q33120) for quantification of DNA.

• Do not use quantification methods such as densitometry (for example, usinga NanoDrop™ spectrophotometer), because they are not specific for DNA.Use of these methods can lead to gross overestimation of the concentrationof sample DNA, under‑seeding of the target amplification reaction, and lowlibrary yields.

2. Normalize each sample of DNA to a final concentration of 3.3 ng/µL (3 µL).

Note: If the DNA concentration is higher than 3.3 ng/µL, dilute the DNA to afinal concentration of 3.3 ng/µL with nuclease‑free water.

Chapter 3 384–well plate format procedureBefore each use of the kit3


Prepare the DNA target amplification reaction

1. Prepare the Amplification Reaction Mix in a 15–mL conical tube.

IMPORTANT! The AgriSeq™ Amplification Mix is viscous. Pipet slowly and mixthoroughly.

Component Volume per well(2X Primer pool)

Volume per well(5X primer pool)

AgriSeq™ Amplification Mix(red cap) 2 µL 2 µL

2X AmpliSeq™ Primer Pool 5 µL —

5X AmpliSeq™Primer Pool — 2 µL

Nuclease-free water — 3 µL

Total Amplification Reaction Mix perwell

7 µL 7 µL


2. Add 7 µL of the Amplification Reaction Mix to each well of a MicroAmp™

EnduraPlate™ Optical 384–Well Clear Reaction Plate (reaction plate).

3. Add 3 µL of normalized DNA to each well containing Amplification ReactionMix. (See the previous section, "Normalize the input DNA".)The total volume for each well is 10 µL.

4. Seal the plate with a MicroAmp™ Clear Adhesive Film, vortex thoroughly, thenbriefly centrifuge to collect contents.


Chapter 3 384–well plate format procedurePrepare the DNA target amplification reaction 3


Amplify the targets

1. Place a MicroAmp™ Optical Film Compression Pad on the reaction plate, thenload the plate in a thermal cycler.

2. Run the following program (10‑µL reaction size).


Hold Activate theenzyme

99°C 2 minutes

Cycle; set numberaccording to thefollowing tables

Denature 99°C 15 seconds

Anneal and extend 60°C 4 or 8 minutes[1]

Hold — 10°C Hold

[1] Use 4 minutes for £1536 primer pairs per pool; 8 minutes for 1,537–5,000 primer pairs per pool.

IMPORTANT! We recommend selecting the number of primer pairs per pool toensure that the mean coverage depth is greater than 100X. There can besignificant marker drop-off when the mean coverage depth is less than 100X. Fordetails, see Appendix B, “Ion Chip™ capacities for AgriSeq™ DNA libraries runon the Ion 540™ Chip“.

Primer pairs per pool (see notes below) Recommended number of amplificationcycles

97–192 18

193–384 17

385–768 16

769–1,536 15

1,537–3,072 14

3,073–5,000 13

STOPPING POINT Target amplification reactions can be stored at 10°C overnighton the thermal cycler. For longer term, store at −20°C.

Chapter 3 384–well plate format procedureAmplify the targets3


Prepare amplicons for barcode addition

1. Prepare the Pre‑ligation Enzyme Reaction Mix in a microcentrifuge tube, thenmix well by pipetting.

Component Volume per well

Pre-ligation Enzyme 1 µL

Nuclease-free Water 1 µL


2. Briefly centrifuge the reaction plate to collect contents. The reaction plate wasprepared in the previous section, "Amplify the targets".

3. Carefully remove the plate seal, then add 2 µL of Pre‑ligation Enzyme ReactionMix (from step 1) to each amplified sample. The total volume per well is 12 µL(10 µL of sample + 2 µL of Pre‑ligation Enzyme Reaction Mix).




Temperature Time

50°C 10 minutes[1]

55°C 10 minutes [1]

60°C 20 minutes

10°C Hold (for up to 1 hour)

[1] Increase to 20 minutes for panels over 1,536 primer pairs/pool.


Chapter 3 384–well plate format procedurePrepare amplicons for barcode addition 3


Add barcodes to the sample libraries

If there is visible precipitate in the Barcoding Buffer or the tube cap after thawing,vortex or pipet up and down at room temperature to resuspend before pipetting.

1. Prepare the Barcode Reaction Mix in a microcentrifuge tube:

Components Volume per well

Barcoding Buffer 2 µL

Barcoding Enzyme 1 µL

Total volume per well 3 µL


2. Briefly centrifuge the reaction plate to collect the contents.

3. Carefully remove the plate seal, then discard.

4. Prepare the IonCode™ Barcode Adapters.a. Briefly centrifuge the IonCode™ Barcode Adapters plate to collect contents.

b. Carefully remove, then discard the plate seal.

5. Add 1 µL of an IonCode™ Barcode Adapter to each sample well of the reactionplate, using a different barcode adapter for each sample.

6. Seal the IonCode™ Barcode Adapters plate with a new MicroAmp™ AdhesiveFilm, then store as recommended.

7. Add 3 µL of the Barcode Reaction Mix to each sample well of the reaction plate.




Temperature Time

22°C 30 minutes

72°C 10 minutes

10°C Hold[1]

[1] Up to 24 hours

STOPPING POINT Libraries can be stored at –20°C.

Chapter 3 384–well plate format procedureAdd barcodes to the sample libraries3


Pool the libraries: 384‑well plate format

IMPORTANT! Pooling can alter coverage depth. There can be significant markerdrop-off when the mean coverage depth is less than 100X. For details, see Appendix B,“Ion Chip™ capacities for AgriSeq™ DNA libraries run on the Ion 540™ Chip“.

1. Briefly centrifuge the plate to collect the contents.

2. Combine 5 µL from each well of a single column of the 384‑well reaction plate(16 samples) into a single well of an 8‑strip tube, then mix well.

4 5 6 7 8 9 10 11 12

The following figure is an example of pooling strategies from a 384‑well plate to a 96‑wellplate.

Note: Alternate strategies can also be used, as long as a total of 16 samples arecombined per pool.

Chapter 3 384–well plate format procedurePool the libraries: 384‑well plate format 3


3. Transfer 60 µL from each well of the 8‑strip tube to a new well on a 96‑well PCRplate (sample plate).

Note:· Four 384–well plates can be loaded and cleaned on the same post‑pooling

plate.· If running fewer than 384 samples, adjust the number of pools accordingly. For

example, if you are running 80 samples, only 5 pools are created.

Prepare Normalization Reaction Mix

Prepare Normalization Reaction Mix in a microcentrifuge tube before AMPure™

library clean‑up, so that the normalization procedure can be started immediately afterpurification of the unamplified libraries, which avoids over‑drying the beads at theend of the purification.

Component Volume per reaction (96–well format)

Normalization Master Mix 100 µL

Normalization Primers 4 µL


Store on ice (or at 4°C) until use.

Purify the unamplified libraries

IMPORTANT!· Bring the AMPure™ XP reagent to room temperature and vortex to disperse the

beads before use. The AMPure™ XP Reagent is purchased separately (see “Requiredmaterials not supplied“ on page 6).

· Pipet the solution slowly.· Use freshly prepared 70% ethanol for the next steps. (Combine 460 µL of 100%

Ethanol with 200 µL of nuclease‑free water per sample.)

1. To each well of the sample plate with the pooled libraries (prepared in step 3, “Pool the libraries: 384‑well plate format“ on page 27), add 90 µL of Agencourt™

AMPure™ XP Reagent, then pipette up and down 5 times to thoroughly mix thebead suspension with the DNA.

Note: Only the wells containing the pooled libraries should be processed.




Chapter 3 384–well plate format procedurePrepare Normalization Reaction Mix3






7. Remove the plate from the magnetic stand, then add 60 µL of Low TE to eachwell of the sample plate with pooled libraries.

8. Pipette up and down 5 times to thoroughly mix.


10. Add 72 µL of the Agencourt™ AMPure™ XP Reagent to each well of the sampleplate with pooled libraries, then pipet up and down 5 times to thoroughly mixthe bead suspension with the DNA.








16. Dry the beads, uncovered, at 40°C for 1 minute. Do not over-dry.

IMPORTANT! Proceed immediately to Normalization. Do not allow the beads tosit uncovered and over‑dry.

Chapter 3 384–well plate format procedurePurify the unamplified libraries 3


Normalize the libraries

• Warm all the AgriSeq™ Normalization Reagents to room temperature.• Vortex and centrifuge all reagents before use.

1. Add 104 µL of Normalization Reaction Mix to each bead pellet. TheNormalization Reaction Mix was prepared in “Prepare Normalization ReactionMix“ on page 28.

2. Seal the plate with a MicroAmp™ Adhesive Film, vortex thoroughly, thencentrifuge to collect contents. Alternatively, mix by pipetting at least half the totalvolume up and down at least 5 times before sealing the plate.

3. Place a MicroAmp™ Optical Film Compression Pad on the plate, then load in thethermal cycler. Run the following program (104‑µL reaction volume).

Note: Library amplification takes place in the presence of the AMPure® XPbeads.


Hold Activate enzyme 98°C 2 minutes

9 cycles Denature 98°C 15 seconds

Anneal/Extend 64°C 1 minute

Hold — 10°C Hold (up to 1hour)

1. After thermal cycling, briefly centrifuge the plate to collect the liquid in the wells.

2. Remove the seal from the plate, then add 20 µL of Normalization Reagent to eachlibrary amplification reaction.

3. Mix by pipetting at least half of the total volume up and down at least 5 times.


1. If not already prepared, wash the Normalization Beads.a. Thoroughly mix the Normalization Beads, transfer 6 µL/reaction to a fresh

1.5–mL microcentrifuge tube, then capture the beads on a magnet for3 minutes.

b. Remove, then discard the supernatant.

c. Add Normalization Wash Buffer to each pellet (12 µL/reaction).

Prepared beads can be stored at 4°C for up to 6 months.

2. Mix the pre‑washed Normalization Beads by gentle vortexing or pipetting upand down.

3. Add 12 µL of washed beads to each well containing the normalization reaction.

Before you begin

Amplify the library

Add NormalizationReagent to theamplified library

Add NormalizationBeads and wash

Chapter 3 384–well plate format procedureNormalize the libraries3


4. Mix by pipetting at least half the total volume up and down at least 5 times.


6. Place the plate in the magnet, then incubate for 2 minutes or until the solution isclear.


8. Add 150 µL of Normalization Wash Buffer to each well containing thenormalization reaction.

9. Place the plate on a magnet, then move the plate side‑to‑side in the two positionsof the magnet to wash the beads.


10. Incubate the plate on the magnet for 2 minutes or until the solution is clear.

11. With the plate still in the magnet, carefully remove, then discard the supernatantwithout disturbing the pellet.

12. Repeat the bead wash as described in step 8–step 11.

IMPORTANT! Ensure that as much wash buffer as possible is removed withoutdisturbing the pellet.

1. Remove the plate from the magnet, then add 100 µL of Normalization ElutionBuffer to each pellet.

2. Seal the plate with MicroAmp™ Clear Adhesive Film, vortex thoroughly, thencentrifuge to collect contents. Alternatively, mix by pipetting at least half the totalvolume up and down at least 5 times before sealing the plate.

Note: Centrifuge with sufficient force to collect droplets, but not to pellet thebeads. If beads are pelleted, vortex again and centrifuge more gently.

3. Elute the library by incubating in a thermal cycler at 32°C for 5 minutes.

4. Place the plate in the magnet, then incubate at room temperature for 5 minutes oruntil the solution is clear.

5. (Optional) To store unpooled libraries, transfer the supernatant to a new well forstorage.

STOPPING POINT

· The supernatant can be stored at 4–8°C for 1 month.· For longer term storage, store libraries at −20°C.

Elute thenormalized library



Final library pooling

Combine 50 µL of each normalized library in a single pooled sample of 1200 µL.

Proceed to template preparation. For details, see the user guide for your templatepreparation kit.

STOPPING POINT

· The supernatant contains the Normalized library at ~200 pM.· Store the remainder of the libraries at 4–8°C for 1 month.· For longer term storage, store libraries at −20°C.

Chapter 3 384–well plate format procedureFinal library pooling3


Troubleshooting

Library yield and quantification

Observation Possible cause Recommended action

Library concentration is low(Library concentration is NOTindicative of quality.)

Sample DNA was mis-quantified.

Requantify sample DNA using the Qubit™

dsDNA HS Assay Kit or the Quant-iT™ DNAAssay Kit, High Sensitivity.

Residual ethanol fromAMPure™ purification inhibitedlibrary amplification.

Carefully remove all drops of ethanol beforelibrary amplification, then centrifuge plate, ifneeded. Repeat residual ethanol removal.

Sample DNA quality was low. Add more DNA or increase target amplificationcycles.

PCR, digestion, or ligation wasinefficient.

Ensure proper dispensing and mixing ofviscous components at each step.

AMPure™ XP Beads were over-dried.

Do not dry the AMPure™ XP Beads more than1 minute.

Normalization Beads were notwashed.

Be sure to wash Normalization Beads beforeuse.

Wrong Normalizations Primerswere used.

Use the Normalization Primers that areprovided in the AgriSeq™ HTS Library Kit.

Residual Normalization Washwas present after wash.

Carefully remove all the Normalization Washbefore elution.

Library concentration is toohigh

Sample DNA or RNA was mis-quantified.

Requantify sample DNA using the Qubit™

dsDNA HS Assay Kit or the Quant-iT™ DNAAssay Kit, High Sensitivity.

More than 10 ng of sample DNAwas used.

Add the recommended amount DNA (10 ng) ordecrease target amplification cycles.

High molecular weight materialis present during sequencing

High molecular weight DNAwas not removed duringpurification of the amplifiedlibrary (does not interfere withsequencing).

No action required.

Inserts form concatemersduring the ligation step.

Reduce nucleic acid input amount.

Requantify sample(s) with the Qubit™ dsDNAHS Assay Kit or the Quant-iT™ DNA Assay Kit,High Sensitivity.

Reduce target amplification cycle number.

A


Low amplicon uniformity (DNA only)


Short amplicons are under-represented Purification was poor. Vortex AMPure™ XP Reagentthoroughly before use, and besure to dispense the fullvolume.

Amplicons were denaturedwhen being prepared for theaddition of barcodes.

Use the 60°C/20 minutetemperature incubation whenpreparing amplicons for theaddition of barcodes.

Long amplicons are under-represented (shortlibrary reads)

Sample DNA is degraded. Repurify the DNA.

PCR was inefficient. Double the anneal and extendtime.

Too few nucleotide flows wereused.

Use an appropriate number offlows to sequence throughamplicons.

Sample was over-treated withPre-ligation Enzyme.

Add no more than 1 µL Pre-ligation Enzyme per 10 µLtarget amplification reaction.

Keep the plate on ice duringPre-ligation Enzyme addition,then transfer to a preheatedthermal cycler immediately.

Denaturation temperaturewas too high.

Use a 97°C denaturationtemperature instead of 99°Cin target amplificationreactions.

AT-rich amplicons are under-represented

Example of loss of AT-rich amplicons. Within theCoverage Analysis Plugin, ampliconrepresentation is plotted by GC content for an IonAmpliSeq™ Panel. Amplicons with 23-50% GCshow an excess failure rate (less than 20% of themean read depth).

Target amplification wasinefficient.

Double the anneal/extendtime in the targetamplification reaction.

Decrease the anneal/extendtemperature of the targetamplification reaction from60°C to 58°C.

Amplicons were denaturedduring preparation for theaddition of barcodes.

Use the 60°C/20 minutetemperature incubation in the"Prepare amplicons forbarcode addition" section.

Amplicons were improperlyprepared for barcodeaddition.

Increase incubation times to20 minutes for each stepduring incubation of the Pre-ligation Enzyme reaction("Prepare amplicons forbarcode addition").

Appendix A TroubleshootingLow amplicon uniformity (DNA only)A



GC-rich amplicons are under-represented

Example of loss of GC-rich amplicons. Within theCoverage Analysis Plugin, ampliconrepresentation is plotted by GC content for an IonAmpliSeq™ Panel. Amplicons with 60-80% GCshow an excess failure rate (less than 20% of themean read depth).

Denaturation was inadequateduring target amplification.

Use a calibrated thermalcycler.

Target amplification wasinefficient.

Increase the anneal/extendtemperature of the targetamplification reaction from60°C to 62°C for the first2 cycles of the targetamplification reaction.

Library amplification wasinefficient.

Do not amplify the library (notrequired for qPCRquantification).

Sample-to-sample representation is not balanced Amount of reaction mix intarget amplification reactionsvaried.

Make a master mix for eachsample DNA.

DNA was not properlynormalized and quantitatedbefore library amplification.

Use Qubit™ dsDNA HS AssayKit or the Quant-iT™ DNAAssay Kit, High Sensitivity toquantitate samples beforelibrary preparation. Ensurethat each sample isnormalized to 3.3 ng/uL(10 ng per reaction) bydiluting samples in nuclease-free water before libraryamplification.

Uniformity is low (without bias) Amplification was inadequate. Double the recommendedanneal/extend time for targetamplification.

Other


Adapter dimers are presentduring sequencing

Adapter dimers. Barcodeadapters run at ≈53 bp, andbarcode adapter dimers run at≈105 bp.

Adapter dimers are formedduring reaction setup or duringdigestion.

Do not combine adapters with the BarcodingReaction Mix before addition.

Use a 65°C temperature incubation instead of60°C during incubation of the Pre-ligationEnzyme reaction (see "Prepare amplicons forbarcode addition").

The number of on-target readsis lower than expected

Unknown. Increase the number of target amplificationcycles by two, or increase the anneal/extendtemperature of the target amplificationreaction from 60°C to 62°C—64°C for the first2 cycles of the target amplification reaction.

Percentage of polyclonal ISPs ishigh (>40%)

Library input was too high. Decrease amount of library added to thetemplate preparation reaction by 50%.

Appendix A TroubleshootingOther A



Percentage of polyclonal ISPs ishigh (>40%)

Library was mis-quantified. Ensure that library was quantified accurately.

Other. Check the appropriate template preparationuser guide for more information.

Low quality ISPs are present athigh percentage (> 15%)

Library input was too low. Double the volume of library used in templatepreparation.

Use a fresh dilution of library prepared in alow-bind tube.

Other. Check the appropriate template preparationuser guide for more information.

Appendix A TroubleshootingOtherA


Ion Chip™ capacities for AgriSeq™

DNA libraries run on the Ion 540™

Chip

The number of combined libraries that can be accommodated in a single sequencingrun depends on the chip, the balance of barcoded library concentration, and thecoverage required.

For a given chip, as the number of amplicons increases, the number of libraries thatcan be accommodated per sequencing run decreases. This relationship is shown in thefollowing table. The numbers in the following table serve as a guide for approximatecapacities. As the number of libraries per chip increases, coverage decreases and itbecomes more difficult to balance the reads between libraries. We suggest combiningfewer libraries initially and determining the real limits empirically.

Number of markers corresponding to the number of barcoded libraries required to achievea mean coverage of 100X.

Barcoded samples Maximum recommended markers[1]

120 5000

192 3125

384 1562

480 1250

576 1041

672 892

768 781

[1] Each marker corresponds to one amplicon.

Note: These recommendations assume 60 million reads using a Ion 540™ chip.

Formula for calculating mean coverage:

Mean Coverage =

[Reads/Chip]

[Number of samples] x [Number of markers]

B


Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, etc). To obtain SDSs, see the “Documentation andSupport” section in this document.

C


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below. Consult the relevant SDSfor specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by thechemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

Appendix C SafetyChemical safety C


Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. Conduct all work in properly equipped facilitieswith the appropriate safety equipment (for example, physical containmentdevices). Safety equipment can also include items for personal protection, suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields, safetyglasses, or goggles. Individuals should be trained according to applicableregulatory and company/ institution requirements before working withpotentially biohazardous materials. Follow all applicable local, state/provincial,and/or national regulations. The following references provide generalguidelines when handling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21‑1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix C SafetyBiological hazard safetyC


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

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• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

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Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact LifeTechnologies at www.thermofisher.com/support.


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http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

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AgriSeq HTS Library Kit MAN0015971 Revision€¦ · Only 12, 24, 36, 48, 60, 72 or 96 samples can...

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