Thermo Fisher Scientific Ion TrueMate Library Preparation User guide

USER GUIDE

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

Ion TrueMate™ Library Preparation

for use with:

Ion TrueMate™ Library Kit

Ion TrueMate™ Plus Library Kit

Catalog Numbers A25614 and A25656

Publication Number MAN0010280

Revision B.0

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

DISCLAIMER

TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,

PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR 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.

This product was developed and manufactured by Lucigen Corporation, Middleton, WI.

Trademarks

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Accura, ChimeraCode, and JunctionCode

are trademarks of Lucigen, used under permission and license. AMPure and Agencourt are trademarks of Beckman Coulter, Inc. Eppendorf LoBind

and Thermomixer are trademarks of Eppendorf AG. Aligent and Bioanalyzer are trademarks of Agilent Technologies, Inc. Hydroshear is a trademark

of Digilab, Inc. Megaruptor is a trademark of Diagenode SA. Pippen Prep is a trademark of Sage Science. DNASTAR, Lasergene, and SeqMan NGen

are trademarks of DNASTAR, Inc.

Contents

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

Revision history ................................................................. 6

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

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

Kit compatibility ................................................................ 7

Kit contents and storage ......................................................... 7

Required materials and equipment ................................................ 9

Procedure guidelines ........................................................... 10

Procedure overview ............................................................ 11

Workflow diagram ............................................................. 12

■CHAPTER 2 Before you begin .......................................... 13

Determine optimal restriction enzyme(s) for digestion .............................. 13

Restriction enzyme options ...................................................... 13

General digestion set-up ........................................................ 14

Example restriction enzyme digests .............................................. 15

■CHAPTER 3 Prepare adapter-compatible DNA ...................... 18

Shear DNA to appropriate size ................................................... 18

Purify sheared DNA ............................................................ 19

Confirm size and quantity of purified sheared DNA ................................. 20

End repair DNA ................................................................ 20

A-tail DNA .................................................................... 21

■CHAPTER 4 Ligate adapters to DNA fragments ..................... 22

Ligate adapters to DNA fragments ............................................... 22

Clean up barcoded DNA ......................................................... 23

Ion TrueMate™ Library Preparation

3

■CHAPTER 5 Size-select DNA and ligate to coupler .................. 25

Size-select barcoded DNA ....................................................... 25

Ligate size-selected DNA to coupler .............................................. 27

Remove linear DNA ............................................................ 28

Bead clean-up ................................................................. 29

■CHAPTER 6 Restriction digest and purify target DNA ............... 30

Digest insert/coupler ........................................................... 30

Biotin-capture to remove unwanted DNA fragments ................................ 31

■CHAPTER 7 Ligate JunctionCode™ sequence and re-

circularize DNA ........................................................... 33

Ligate JunctionCode™ sequence to biotin-captured insert/coupler ................... 33

Clean up JunctionCode™-ligated DNA ............................................. 34

Re-circularize DNA ............................................................. 35

Remove unwanted linear DNA ................................................... 36

Clean up exonuclease-treated insert/coupler ...................................... 36

■CHAPTER 8 Amplify DNA ............................................... 38

Identify optimal amplification conditions .......................................... 38

Generate amplified library ...................................................... 40

■CHAPTER 9 Size select library ........................................ 42

Purify amplified library ......................................................... 42

(Recommended)

Size select library using Pippin Prep™............................. 43

Purify mate-pair library .................................................... 43

(Optional)

Size select library using AMPure® beads ................................ 44

First-round purification .................................................... 45

Second-round purification .................................................. 45

■CHAPTER 10 Sequence data analysis ................................. 48

Ion PGM™ sequence data analysis ................................................ 48

■APPENDIX A Troubleshooting ......................................... 49

■APPENDIX B Restriction enzyme choice ............................. 51

Mate-tag size depends on restriction enzyme choice ............................... 51

Contents

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Ion TrueMate™ Library Preparation

■APPENDIX C DNA size selection vs. insert size ...................... 52

DNA size selection vs. insert size ................................................ 52

■APPENDIX D Determining bead concentration ...................... 53

Determining bead concentration for size selection ................................. 53

■APPENDIX E Sequence analysis ....................................... 54

Overview ...................................................................... 54

Software requirements ......................................................... 54

Sequence analysis example ..................................................... 55

Assembly software options ...................................................... 55

■APPENDIX F Sequence and location information .................... 57

Barcode adapters and JunctionCode™ Sequence ................................... 57

ChimeraCode™ Sequences ...................................................... 57

■APPENDIX G Additional test reactions for library amplification ... 59

Additional test reactions for library amplification .................................. 59

■Safety ...................................................................... 63

Chemical safety ................................................................ 64

Biological hazard safety ........................................................ 65

■Documentation and support ............................................. 66

Obtaining SDSs ................................................................ 66

Obtaining Certificates of Analysis ................................................ 66

Limited product warranty ....................................................... 66

Customer and technical support ................................................. 66

Contents

Ion TrueMate™ Library Preparation

5

About this guide

IMPORTANT! Before using this product, read and understand the information in the

“Safety” appendix in this document.

Revision history

Revision Date Description

A.0 25 July 2014 • New user guide

B.0 12 March 2015 • Revised software scripts

• Revised software example

6

Ion TrueMate™ Library Preparation

Product information

Product description

The Ion TrueMate™ Library Kit and Ion TrueMate™ Plus Library Kit are designed to

generate mate pair libraries, with inserts 2–8 Kb in size, for sequencing on the Ion

PGM™ System.

Kit compatibility

Ion TrueMate™ Library kits (Cat. nos. A25614 and A25656) are compatible with the

Ion PGM™ Template OT2 400 Kit (Cat. no. 4479878), Ion PGM™ Sequencing 400 Kit

(Cat. no. 4482002), and Ion 318™ Chip Kit v2 (Cat. no. 4484354) for use with the Ion

PGM™ System.

Kit contents and storage

Ion TrueMate™ Library Kit (Cat. no. A25614) and Ion TrueMate™ Plus Library Kit

(Cat. no. A25656) include the following:

Ion TrueMate™ End Repair Kit[1]

Component Cap color Quantity Volume Storage

Elution Buffer clear 1 520 µL

–30°C to–10°C

End Repair Tailing

Buffer red 2 1 mL

End Repair Enzyme

Mix red 1 160 µL

Klenow Fragment violet 1 160 µL

Barcode A white 1 480 µL

Ligase blue 1 320 µL

1

Ion TrueMate™ Library Preparation

7

Ion TrueMate™ Library Reagents[2]

Component Cap color Quantity Volume Storage

Elution Buffer clear 5 1.6 mL

–30°C to–10°C

Klenow Fragment violet 1 60 µL

Ligase blue 1 105 µL

Mate Pair Coupler

Mix red 1 30 µL

10X Ligase Buffer blue 1 600 µL

Nuclease I black 1 100 µL

Nuclease II black 1 75 µL

Biotin Wash Buffer yellow 4 1.5 mL

Biotin Capture

Buffer yellow 1 450 µL

Biotin Capture

Reagent yellow 1 15 µL

Biotin Elution

Buffer yellow 1 520 µL

Tailing Buffer orange 1 500 µL

JunctionCode™

Sequence white 1 60 µL

T4 Polynucleotide

Kinase green 1 20 µL

Accura™ Hot Start

2X Master Mix brown 1 1.3 mL

Amplification

Primer Mix brown 1 260 µL

[1] May be ordered separately (Part. no. A25655).

[2] May be ordered separately (Part. no. A25652).

In addition, the Ion TrueMate™ Plus Library Kit (Cat. no. A25656) also includes:

Ion TrueMate™ RE Kit[1, 2]

Component Cap color Quantity Volume Storage

Rsa

I (10 U/µL) red 1 tube 30 µL

–30°C to–10°C

Alu

I (10 U/µL) orange 1 tube 30 µL

Hae

III (10 U/µL) yellow 1 tube 30 µL

Acc

II (10 U/µL) green 1 tube 30 µL

Chapter 1 Product information

Kit contents and storage

1

8

Ion TrueMate™ Library Preparation

Ion TrueMate™ RE Kit[1, 2]

Component Cap color Quantity Volume Storage

10X RE Buffer –30°C to–10°Cwhite 1 tube 120 µL

[1] May be ordered separately (Part. no. A25653).

[2] Thaw all kit reagents on ice prior to use.

Required materials and equipment

Unless otherwise specified, all materials are available from Life Technologies

(www.lifetechnologies.com). MLS: Fisher Scientific (www.fisherscientific.com) or

major laboratory supplier.

Component Supplier Cat. No. Quantity

(Optional)

HpyCH4V

Restriction Enzyme New England

Biolabs R0620S 1

10X CutSmart™ Buffer

Dynabeads® MyOne™

Streptavidin C1 Life Technologies 65001 1

Agencourt® AMPure®

XP Magnetic Beads Beckman Coulter A63882 1

100% Ethanol MLS — —

Nuclease-Free Water Ambion AM9930, AM9932,

AM9937,

AM9938, or AM9939

—

1.5-mL Eppendorf

LoBind® Tubes Eppendorf 022431021 1

0.2-mL thin wall PCR

tubes Eppendorf 951010006 1

Qubit® dsDNA BR

Assay Kit Life Technologies Q32850 1

Agilent® High

Sensitivity DNA Kit Agilent 5067-4626 1

(Optional)

DNA 12000

Kit Agilent 5067-1508 1

Qubit® 2.0 Fluorometer

or equivalent Life Technologies Q32866 1

Thermomixer® R Eppendorf 22670107 1

Heat blocks (25°C –

80°C) MLS — 3

Chapter 1 Product information

Required materials and equipment

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Ion TrueMate™ Library Preparation

9

Component Supplier Cat. No. Quantity

DynaMag™-2 Magnet Life Technologies 12321D 1

One of the following:

• GeneAmp® PCR

System 9700

Single or Dual 96-

well Thermal

Cycler

• AB® 2720 Thermal

Cycler

• Veriti® 96-well

Thermal Cycler

• ProFlex™ 96-Well

PCR System

Life Technologies See web product

pages 1

Microcentrifuge (for

quick ~ 2000 x g spins) MLS — 1

Agilent® 2100

Bioanalyzer®

Instrument

Agilent G2939AA 1

Sage Pippin Prep™Sage Science See web product

pages 1

Pippin Prep™ 1.5%

Agarose Cassettes Sage Science CDF 1510 —

Electrophoresis

equipment and

reagents or E‑Gel®

system

— — —

One of the following:

• HydroShear®

• Megaruptor®

• Covaris® g-TUBE

• Digilab Inc.

• Diagenode Inc.

• Covaris

See web product

pages

520079 or 520104

1

Procedure guidelines

• Use good laboratory practices to minimize cross-contamination of products. If

possible, perform library construction in an area or room that is distinct from that

of template preparation.

• Perform all steps requiring 1.5 mL tubes with 1.5 mL Eppendorf LoBind® tubes

(Eppendorf® Cat. no. 022431021).

• Thaw reagents on ice before use, and keep enzymes at –30°C to –10°C until ready

to use.

• Mix reagents thoroughly before use, especially if frozen and thawed.

Chapter 1 Product information

Procedure guidelines

1

10

Ion TrueMate™ Library Preparation

Procedure overview

Using this method, genomic DNA is sheared to 400–500 bp fragments, end-repaired,

A-tailed, and ligated to barcode adapters. The inserts are size-selected and ligated to a

unique coupler that contains encrypted ChimeraCode™ sequences that help identify

true mates, minimizing false mate pair fragment configurations. The circularized

inserts are then treated with exonuclease to remove unwanted linear DNA, and then

digested with a selection of endonucleases to produce the correctly sized mate-tags

(di-tags). Biotin capture is used to remove the unwanted DNA fragments prior to the

addition of a JunctionCode™ sequence that identifies mate pair fragment junctions.

The library is re-circularized and amplified by PCR, prior to template preparation and

sequencing on the Ion PGM™ System.

Chapter 1 Product information

Procedure overview

1

Ion TrueMate™ Library Preparation

11

Workflow diagram

Figure 1: Schematic of Ion TrueMate™ library construction.

Chapter 1 Product information

Workflow diagram

1

12

Ion TrueMate™ Library Preparation

Before you begin

Determine optimal restriction enzyme(s) for digestion

Note: The procedure described in this section is designed to determine the optimal

restriction enzymes to use in “Digest insert/coupler“ on page 30.

Before proceeding with library construction, the restriction enzyme(s) needed to

digest the gDNA to 400–500 bp (desired final library after PCR amplification) must be

identified. This step is critical to ensure the kit performs as designed and the

sequencing coverage is uniform.

The restriction enzyme(s) needed will vary for each genome. The optimal digestion

method may be multiple digests with individual restriction enzymes or multiple

digests with a combination of two or more restriction enzymes. Each enzyme or

combination of enzymes will produce a different digestion pattern and will add

diversity to the genome coverage.

We recommend performing multiple digestion reactions using both individual

restriction enzymes and combinations of enzymes provided in the Ion TrueMate™ RE

Kit, and then visualize the digest results on an E-Gel®, Agilent® Bioanalyzer®, or

agarose gel. Start by digesting with the four individual restriction enzymes. After

visualizing these digests on a gel, subsequent combination digests can be tested. For

example, two or more infrequent cutting enzymes can be combined to produce the

desired digestion pattern. The four included restriction enzymes will likely be

sufficient to obtain desired results; however, if you require additional ones, we

recommend HpyCH4V (New England Biolabs, Cat. no. R0626S). Example gels using

both the provided enzymes as well as HpyCH4V are included in “Example restriction

enzyme digests“ on page 15.

See Appendix B, “Restriction enzyme choice“, for additional information on the

impact of the restriction enzyme choice on the final library.

Restriction enzyme options

Enzyme Source

Rsa

I (10U/µL) Ion TrueMate™ RE Kit

Alu

I (10U/µL)

Hae

III (10U/µL)

Acc

II (10U/µL)

HpyCH4V

(5U/µL) New England Biolabs

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Ion TrueMate™ Library Preparation

13

General digestion set-up

1. Add the following reagents to 0.2 mL thin wall PCR tube(s):

Reagent Final amount per reaction

gDNA 200 ng

Buffer[1] 1X

Restriction enzyme(s)[2,3] 10 Units[4]

Nuclease-Free Water to 40 µL

Total 40 µL

[1] Use of 10X RE Buffer (supplied in Ion TrueMate™ RE Kit) or CutSmart Buffer (NEB) is highly

recommended. Buffers from other vendors have not been tested and may not be compatible with the

restriction enzyme(s) used to digest the gDNA.

[2] If using a pool of restriction enzymes, mix 10 Units of each enzyme in a 1.5 mL LoBind® tube and use

1 µL for the digest.

[3]

HpyCH4V

can be used in either 10X RE Buffer (supplied in Ion TrueMate™ RE Kit) or CutSmart Buffer

(NEB).

[4] Not to exceed 4 µL.

2. Mix by pipetting up and down 10 times.

3. Incubate reaction(s) at 37°C for 30 minutes.

4. Run reaction(s) alongside undigested gDNA on 1.7% agarose gel with DNA

ladder (e.g. 100 bp ladder).

5. Review results and determine optimal restriction enzyme(s).

Note: Ideal digests will contain the majority of the smear within 400–500 bp size

range. For optimal sequencing results, perform two restriction digests using at

least one enzyme for each digest and pool the digested material prior to cleanup

(see figures 2, 3, and 4).

The table below provides recommendations for restriction enzymes for three

reference genomes:

Genome GC content Restriction enzymes

E. coli

(DH10B) 50% Reaction 1:

HpyCH4V

Reaction 2:

Acc

II

T. aquaticus

68% Reaction 1:

Alu

I

Reaction 2:

Rsa

I +

Acc

II

H. sapiens

50% Reaction 1:

Alu

I

Reaction 2:

Hae

III +

Rsa

I

Chapter 2 Before you begin

General digestion set-up

2

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Ion TrueMate™ Library Preparation

Example restriction enzyme digests

The gel images below provide examples of restriction enzyme testing that were used

to identify recommended restriction enzymes:

Figure 2: Results of E. coli restriction digest testing. Optimal results are seen with

HpyCH4V and Acc II.

Chapter 2 Before you begin

Example restriction enzyme digests

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Ion TrueMate™ Library Preparation

15

Figure 3: Results of T. aquaticus restriction digest testing. Optimal results are seen

with AluI and RsaI +AccII.

Chapter 2 Before you begin

Example restriction enzyme digests

2

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Ion TrueMate™ Library Preparation

Figure 4: Results of human gDNA restriction digest testing. Optimal results are seen

with AluI and HaeIII + RsaI .

Chapter 2 Before you begin

Example restriction enzyme digests

2

Ion TrueMate™ Library Preparation

17

Prepare adapter-compatible DNA

Shear DNA to appropriate size

During this step, the genomic DNA (gDNA) is sheared to an average size range that is

larger than the desired insert size. See Appendix B, “DNA size selection vs. insert

size“ on page 52 for additional information on shearing and size selection.

Note: gDNA used must be free of contaminating RNA.

A sample loss of 20–50% is expected during shearing and bead clean up. The

percentage of sample loss will vary depending on the shearing method used. This

expected loss should be taken into account when determining the amount of gDNA to

shear.

The table below provides examples of recommended shearing size, amount of starting

gDNA, and shearing method for various libraries:

Desired

insert size

Sheared

fragment

size

Recommended

amount of starting

material Recommended shearing method[1]

2 Kb 3 Kb 2.5 – 3 µg 3 Kb setting with Megaruptor® short

hydropore[2]

5 Kb 6 Kb 3.5 – 5 µg 8 Kb setting with Covaris® g- TUBES or

Megaruptor®

8 Kb 10 Kb 11.5 –16 µg 10 Kb setting with Covaris® g- TUBES or

Megaruptor®

[1] Use manufacturer's recommended protocols.

[2] Higher sample loss (up to 50 or 60%) is expected using the Megaruptor®.

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Ion TrueMate™ Library Preparation

Purify sheared DNA

The following protocol describes how to perform a bead clean-up to concentrate and

purify the sheared DNA from the previous step.

IMPORTANT! Reagents used from this point through "Ligate adapters to DNA

fragments" are included in the End Repair Kit (Cat. no. A25655).

1. Transfer the sheared gDNA into a 1.5 mL LoBind® tube.

2. Add an equal volume AMPure® XP beads to the sheared gDNA at room

temperature to obtain a 1:1 ratio of beads to sample.

3. Mix the beads and gDNA gently by pipetting up and down 10 times.

4. Incubate the mixture at room temperature for 5 minutes. Do not keep the tube in

the magnetic rack during incubation.

5. Pulse-spin to bring the contents to the bottom of the tube. Place in the magnetic

rack for 5 minutes, or until the solution becomes clear.

6. Carefully remove and discard the supernatant with a pipet, without disturbing

the bead pellet.

7. Without removing the tube from the magnet, add 750 μL of freshly prepared

70% ethanol.

8. Incubate for 30 seconds, turning the tube around twice in the magnet to move the

beads around. After the solution clears, remove and discard the supernatant

without disturbing the pellet.

9. Repeat step 7 and 8 for a second wash.

10. Pulse-spin the tube, place it back in the magnetic rack, and remove any

remaining residual ethanol with a 20-μL pipettor, without disturbing the pellet.

11. Keeping the tube on the magnet, air-dry the beads at room temperature for

~5 minutes.

12. Remove the tube from the magnetic rack, and add 52 μL of Elution Buffer (from

End Repair Kit) directly to the pellet to disperse the beads.

13. Mix thoroughly by pipetting the suspension up and down 5 times. Do not vortex

the tube.

14. Pulse-spin and place the tube in the magnetic rack for at least one minute until

the solution clears.

15. Transfer 50 μL of the supernatant containing the sheared DNA to a new 1.5 mL

LoBind® tube for size confirmation and quantitation.

Chapter 3 Prepare adapter-compatible DNA

Purify sheared DNA

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Ion TrueMate™ Library Preparation

19

Confirm size and quantity of purified sheared DNA

1. Confirm the size of the sheared gDNA by visualization on a 0.8% E-Gel®,

Agilent® Bioanalyzer® with the DNA 12000 Kit, or agarose gel. See Appendix C,

“DNA size selection vs. insert size“, panel C, for example images of sheared

gDNA.

2. Quantify the sample using the Qubit® dsDNA BR Assay Kit with the Qubit® 2.0

Fluorometer according to manufacturer's instructions.

The following table provides information about minimum amounts and

concentrations of DNA required to proceed:

Insert size Minimum amount of

DNA required Minimum concentration of DNA

required (after elution)

up to 2 Kb 1.0 µg ³22 ng/µL

> 2–5 Kb 2.5 µg ³50 ng/µL

>5–8 Kb 8 µg ³160 ng/µL

STOPPING POINT DNA may be stored at 10°C overnight. For longer periods, store

at –20°C.

End repair DNA

During this step, the purified, sheared gDNA is end repaired. Each end-repair

reaction is limited by the number of DNA molecules. Therefore, the number of

reactions performed at this step is determined by the insert size:

Insert size Recommended number of reactions

Up to 2 Kb 2

> 2–5 Kb 5

> 5–8 Kb 8

1. Aliquot gDNA into 0.2-mL thin-wall PCR tubes, then add the following

components to each tube:

Component Amount per

reaction (< 5 Kb

inserts)

Amount per

reaction (³ 5–8 Kb

inserts)

Purified, sheared gDNA 500 ng 1 µg

End Repair Tailing Buffer (red cap) 25 µL 25 µL

End Repair enzyme Mix (red cap) 2 µL 2 µL

Nuclease-Free Water up to 23 µL up to 23 µL

Total 50 µL 50 µL

2. Mix by pipetting up and down 10 times.

Chapter 3 Prepare adapter-compatible DNA

Confirm size and quantity of purified sheared DNA

3

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Ion TrueMate™ Library Preparation

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Thermo Fisher Scientific Ion TrueMate Library Preparation User guide

Thermo Fisher Scientific Ion TrueMate Library Preparation User guide

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