TruSeq DNA PCR-Free Library Prep Protocol Guide (15075699 A)

TruSeq DNA PCR-Free Library Prep Protocol Guide (15075699 A)

TruSeq DNA PCR-Free Library Prep

Protocol Guide

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

Fragment DNA

Repair Ends and Select Library Size

Adenylate 3ʹ Ends

Ligate Adapters

Validate Libraries

Normalize and Pool Libraries

Acronyms

Technical Assistance

10

11

12

13

7

8

3

5

ILLUMINA PROPRIETARY

Part # 15075699 Rev. A

June 2015

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.

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MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND

DAMAGE TO OTHER PROPERTY.

ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)

DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).

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Fragment DNA

Procedure

1 Quantify gDNA using a fluorometric-based method.

2 Normalize gDNA with RSB to 55 µl in the DNA plate.

• 20 ng/µl for a 350 bp insert size

• 40 ng/µl for a 550 bp insert size

3 Mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

4 Centrifuge as follows.

• [HS] Centrifuge at 280 × g for 1 minute.

• [LS] Centrifuge briefly.

5 Transfer 52.5 µl DNA to Covaris tubes.

6 Centrifuge at 280 × g for 5 seconds.

7 Fragment the DNA using the following Covaris settings.

Table 1 350 bp Insert Settings

Covaris Setting

Duty Factor (%)

Intensity

Peak/Displayed Power (W)

Cycles/Burst

Duration (seconds)

Mode

Temperature (°C)

M220

20

50

65

20

S220 S2 E210

5 10

175 23

5.0

200

50 45

Frequency sweeping

5.5–6

14

Table 2 550 bp Insert Settings

Covaris Setting

Duty Factor (%)

Intensity

Peak/Displayed Power (W)

Cycles/Burst

Duration (seconds)

Mode

Temperature (°C)

M220

20

50

45

20

S220 S2 E210

5

175 9

10

2.0

200

25 45

Frequency sweeping

5.5–6

7

8 Centrifuge at 280 × g for 5 seconds.

9 Transfer 50 µl supernatant to the CSP plate.

10 Add 80 µl SPB, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

11 Incubate at room temperature for 5 minutes.

12 [HS] Centrifuge at 280 × g for 1 minute.

13 Place on a magnetic stand and wait until the liquid is clear (~8 minutes).

TruSeq DNA PCR-Free Library Prep Protocol Guide

3

14 Remove and discard all supernatant.

15 Wash 2 times with 200 µl 80% EtOH.

16 Use a 20 µl pipette to remove residual EtOH.

17 Air-dry on the magnetic stand for 5 minutes.

18 Add 52.5 µl RSB.

19 Remove from the magnetic stand, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

20 Incubate at room temperature for 2 minutes.

21 [HS] Centrifuge at 280 × g for 1 minute.

22 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).

23 Transfer 50 µl supernatant to the IMP plate.

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Part # 15075699 Rev. A

Repair Ends and Select Library Size

Preparation

1 [HS] Preheat the microheating system to 30°C.

2 [LS] Save the following ERP program on the thermal cycler:

• Choose the preheat lid option and set to 100°C

• 30°C for 30 minutes

• Hold at 4°C

Procedure

1 Add 10 µl CTE.

2 Add 40 µl ERP2 or ERP3, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

3 [HS] Centrifuge at 280 × g for 1 minute.

4 Incubate as follows.

• [HS] Place on the 30°C microheating system with the lid closed for 30 minutes, and then place on ice.

• [LS] Place on the thermal cycler and run the ERP program.

5 Dilute SPB with PCR grade water to 160 µl per 100 µl of end-repaired sample.

• When processing ≤ 6 samples, use a new 1.7 ml microcentrifuge tube.

• When processing > 6 samples, use a new 15 ml conical tube.

Determine the volumes using the following formulas, which include 15% excess for multiple samples.

Table 3 Diluted SPB for a 350 bp Insert Size

Formula

SPB

PCR grade water

# of samples X 109.25 µl

# of samples X 74.75 µl

Example Amount per 12 samples

1311 µl

897 µl

Your Calculation

Table 4 Diluted SPB for a 550 bp Insert Size

Formula

SPB

PCR grade water

# of samples X 92 µl

# of samples X 92 µl

Example Amount per 12 samples

1104 µl

1104 µl

Your Calculation

6 Vortex diluted SPB until well-dispersed.

7 Add 160 µl diluted SPB, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

8 Incubate at room temperature for 5 minutes.

9 [HS] Centrifuge at 280 × g for 1 minute.

10 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).

11 Transfer 250 µl supernatant to the CEP plate.

TruSeq DNA PCR-Free Library Prep Protocol Guide

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6

12 Add 30 µl undiluted SPB, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

13 Incubate at room temperature for 5 minutes.

14 [HS] Centrifuge at 280 × g for 1 minute.

15 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).

16 Remove and discard all supernatant.

17 Wash 2 times with 200 µl 80% EtOH.

18 Use a 20 µl pipette to remove residual EtOH.

19 Air-dry on the magnetic stand for 5 minutes.

20 Add 17.5 µl RSB.

21 Remove from the magnetic stand, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

22 Incubate at room temperature for 2 minutes.

23 [HS] Centrifuge at 280 × g for 1 minute.

24 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).

25 Transfer 15 µl supernatant to the ALP plate.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

Part # 15075699 Rev. A

Adenylate 3ʹ Ends

Preparation

1 [HS] Preheat 2 microheating systems, the first to 37°C and the second to 70°C.

2 [LS] Save the following ATAIL70 program on the thermal cycler:

• Choose the preheat lid option and set to 100°C

• 37°C for 30 minutes

• 70°C for 5 minutes

• 4°C for 5 minutes

• Hold at 4°C

Procedure

1 Add 2.5 µl CTA.

2 Add 12.5 µl ATL or ATL2, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

3 Incubate as follows.

[HS] a Place on the 37°C microheating system with the lid closed for 30 minutes.

b Move to the 70°C microheating system with the lid closed for 5 minutes.

c Place on ice for 5 minutes.

[LS] a Place on the thermal cycler and run the ATAIL70 program.

TruSeq DNA PCR-Free Library Prep Protocol Guide

7

Ligate Adapters

Preparation

1 [HS] Preheat a microheating system to 30°C.

2 [LS] Save the following LIG program on the thermal cycler:

• Choose the preheat lid option and set to 100°C

• 30°C for 10 minutes

• Hold at 4°C

Procedure

1 Add the following reagents in the order listed, and then mix thoroughly as follows.

Reagent

CTL

LIG2

DNA adapters

Volume (µl)

2.5

2.5

2.5

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

2 Centrifuge at 280 × g for 1 minute.

3 Incubate as follows.

• [HS] Place on the 30°C microheating system with the lid closed for 10 minutes, and then place on ice.

• [LS] Place on the thermal cycler and run the LIG program.

4 Add 5 µl STL, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

5 [HS] Centrifuge at 280 × g for 1 minute.

6 Perform steps

6a

through

6m

using the Round 1 volumes.

a Add SPB, and then mix thoroughly as follows.

SPB

Round 1

42.5 µl

Round 2

50 µl

— [HS] Shake at 1800 rpm for 2 minutes.

— [LS] Pipette up and down.

b Incubate at room temperature for 5 minutes.

c [HS] Centrifuge at 280 × g for 1 minute.

d Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).

e Remove and discard all supernatant.

f Wash 2 times with 200 µl 80% EtOH.

g Use a 20 µl pipette to remove residual EtOH.

h Air-dry on the magnetic stand for 5 minutes.

i Add RSB.

RSB

Round 1

52.5 µl

Round 2

22.5 µl

8

Part # 15075699 Rev. A

j Remove from the magnetic stand, and then mix thoroughly as follows.

— [HS] Shake at 1800 rpm for 2 minutes.

— [LS] Pipette up and down.

k Incubate at room temperature for 2 minutes.

l [HS] Centrifuge at 280 × g for 1 minute.

m Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).

7 Transfer 50 µl supernatant to the CAP plate.

8 Repeat steps

6a

through

6m

with the new plate using the Round 2 volumes.

9 Transfer 20 µl supernatant to the TSP1 plate.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

TruSeq DNA PCR-Free Library Prep Protocol Guide

9

Validate Libraries

1 Quantify the libraries using qPCR, with the following modifications.

}

Use at least 2 µl of the original library stock.

} Perform 2 additional dilutions.

The concentration of each library is calculated as indicated in

Table 5

Table 6 .

Table 5 350 bp Library Concentration Calculation

Dilution

Factor

Calculated by qPCR instrument (pM)*

Average diluted library

(pM)

Size adjusted diluted library (pM)

1:10,000

1:20,000

A1

B1

A2

B2

A =

(A1 + A2)/2

B =

(B1 + B2)/2

W1 =

A x (452/470)

W2 =

B x (452/470)

Undiluted library (pM)

*

C1 =

W1 x 10,000

C2 =

W2 x 20,000

Undiluted library

(pM)

(C1 + C2)/2

Table 6 550 bp Library Concentration Calculation

Dilution

Factor

1:10,000

1:20,000

Calculated by qPCR instrument (pM)*

C1

D1

C2

D2

Average diluted library

(pM)

C =

(C1 + C2)/2

D =

(D1 + D2)/2

Size adjusted diluted library (pM)

W3 =

C x (452/670)

W4 =

D x (452/670)

*Duplicate data points

}

Determine the concentration of the diluted library.

}

Perform a size adjustment calculation.

Undiluted library (pM)

*

C3 =

W3 x 10,000

C4 =

W4 x 20,000

• For 350 bp libraries, use 470 bp for the average fragment length

• For 450 bp libraries, use TBD bp for the average fragment length

Undiluted library

(pM)

(C3 + C4)/2

• For 550 bp libraries, use 670 bp for the average fragment length

}

Calculate the concentration of the undiluted library.

} If a replicate is an outlier, it can be omitted from the calculation. If multiple replicates are outliers, repeat the assay.

Quality Control

Verify fragment size by checking the library size distribution.

1 Dilute the DNA library 1:5 with water.

2 Run 1 µl diluted DNA library on a High Sensitivity DNA chip.

10

Part # 15075699 Rev. A

Normalize and Pool Libraries

Procedure

1 Transfer 5 µl library to the DCT plate.

2 Normalize with Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20 to 2 nM, and then mix thoroughly as follows.

• [HS] Shake at 1000 rpm for 2 minutes.

• [LS] Pipette up and down.

3 [HS] Centrifuge at 280 × g for 1 minute.

4 If pooling 2–24 samples, transfer 5 µl of each normalized library to a single well of the PDP plate.

5 If pooling 25–96 samples, do the following.

a Transfer 5 µl of each column of normalized library to column 1 of the PDP plate, and then mix thoroughly as follows.

— [HS] Shake at 1800 rpm for 2 minutes.

— [LS] Pipette up and down.

b [HS] Centrifuge at 280 × g for 1 minute.

c Transfer the contents of each well of column 1 to well A2.

6 Mix thoroughly as follows.

• [HS] Shake plate at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

7 [HS] Centrifuge at 280 × g for 1 minute.

8 Proceed to cluster generation.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C.

TruSeq DNA PCR-Free Library Prep Protocol Guide

11

Acronyms

Acronym

ALP

ATL

CAP

CEP

CSP

CTA

PDP

RSB

SPB

STL

TSP1

CTE

CTL

DAP

DCT

DNA

ERP

IMP

LIG

Definition

Adapter Ligation Plate

A-Tailing Mix

Clean Up ALP Plate

Clean Up End Repair Plate

Clean Up Sheared DNA Plate

A-Tailing Control

End Repair Control

Ligation Control

DNA Adapter Plate

Diluted Cluster Template Plate

Customer Sample DNA Plate

End Repair Mix

Insert Modification Plate

Ligation Mix

Pooled Dilution Plate

Resuspension Buffer

Sample Purification Beads

Stop Ligation Buffer

Target Sample Plate 1

12

Part # 15075699 Rev. A

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 7 Illumina General Contact Information

Website

www.illumina.com

Email

[email protected]

Table 8 Illumina Customer Support Telephone Numbers

Region

North America

Australia

Austria

Belgium

Denmark

Finland

France

Germany

Ireland

Contact Number

1.800.809.4566

1.800.775.688

0800.296575

0800.81102

80882346

0800.918363

0800.911850

0800.180.8994

1.800.812949

Region

Italy

Netherlands

New Zealand

Norway

Spain

Sweden

Switzerland

United Kingdom

Other countries

Contact Number

800.874909

0800.0223859

0800.451.650

800.16836

900.812168

020790181

0800.563118

0800.917.0041

+44.1799.534000

Safety Data Sheets

Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html

.

Product Documentation

Product documentation in PDF is available for download from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature.

TruSeq DNA PCR-Free Library Prep Protocol Guide

Illumina

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America) [email protected]

www.illumina.com

*15075699*

Part # 15075699 Rev. A

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