TruSeq Nano DNA Libary Prep Protocol Guide (15075697 A)

TruSeq Nano DNA Libary Prep Protocol Guide (15075697 A)
TruSeq Nano DNA 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
Enrich DNA Fragments
Validate Libraries
Normalize and Pool Libraries
Acronyms
Technical Assistance
ILLUMINA PROPRIETARY
Part # 15075697 Rev. A
June 2015
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Fragment DNA
Fragment DNA
Procedure
1
Quantify gDNA using a fluorometric-based method.
2
Normalize gDNA with RSB to 52.5 µl in the DNA plate.
• 100 ng for a 350 bp insert size
• 200 ng 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)
Table 2 550 bp Insert Settings
Covaris Setting
Duty Factor (%)
Intensity
Peak/Displayed Power (W)
Cycles/Burst
Duration (seconds)
Mode
Temperature (°C)
M220
S220
20
—
50
5
—
175
S2
E210
10
5.0
23
14
200
65
—
20
50
45
Frequency sweeping
5.5–6
M220
S220
20
—
50
5
—
175
S2
E210
10
2.0
9
7
200
45
—
20
8
Centrifuge at 280 × g for 5 seconds.
9
Transfer 50 µl supernatant to the CSP plate.
25
45
Frequency sweeping
5.5–6
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 Nano DNA Library Prep Protocol Guide
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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 62.5 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 60 µl supernatant to the IMP plate.
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Part # 15075697 Rev. A
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 40 µl ERP2 or ERP3, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
2
[HS] Centrifuge at 280 × g for 1 minute.
3
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.
4
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
Example Amount
per 12 samples
1311 µl
897 µl
# of samples X 109.25 µl
# of samples X 74.75 µl
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
Your Calculation
5
Vortex diluted SPB until well-dispersed.
6
Add 160 µl diluted SPB, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
7
Incubate at room temperature for 5 minutes.
8
[HS] Centrifuge at 280 × g for 1 minute.
9
Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
10 Transfer 250 µl supernatant to the CEP plate.
TruSeq Nano DNA Library Prep Protocol Guide
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Repair Ends and Select Library Size
Repair Ends and Select Library Size
11 Add 30 µl undiluted SPB, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
12 Incubate at room temperature for 5 minutes.
13 [HS] Centrifuge at 280 × g for 1 minute.
14 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
15 Remove and discard all supernatant.
16 Wash 2 times with 200 µl 80% EtOH.
17 Use a 20 µl pipette to remove residual EtOH.
18 Air-dry on the magnetic stand for 5 minutes.
19 Add 20 µl RSB.
20 Remove from the magnetic stand, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
21 Incubate at room temperature for 2 minutes.
22 [HS] Centrifuge at 280 × g for 1 minute.
23 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
24 Transfer 17.5 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.
6
Part # 15075697 Rev. A
Adenylate 3ʹ Ends
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 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.
2
Centrifuge at 280 × g for 1 minute.
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.
b Centrifuge at 280 × g for 1 minute.
TruSeq Nano DNA Library Prep Protocol Guide
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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
RSB
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
b
c
d
e
f
g
h
i
Round 2
50 µl
— [HS] Shake at 1800 rpm for 2 minutes.
— [LS] Pipette up and down.
Incubate at room temperature for 5 minutes.
[HS] Centrifuge at 280 × g for 1 minute.
Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
Remove and discard all supernatant.
Wash 2 times with 200 µl 80% EtOH.
Use a 20 µl pipette to remove residual EtOH.
Air-dry on the magnetic stand for 5 minutes.
Add RSB.
RSB
8
Round 1
42.5 µl
Round 1
52.5 µl
Round 2
27.5 µl
Part # 15075697 Rev. A
Ligate Adapters
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 25 µl supernatant to the PCR 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 Nano DNA Library Prep Protocol Guide
9
Enrich DNA Fragments
Preparation
1
Save the following PCRNano program on the thermal cycler:
• Choose the preheat lid option and set to 100°C
— 95°C for 3 minutes
• 8 cycles of:
— 98°C or 20 seconds
— 60°C for 15 seconds
— 72°C for 30 seconds
• 72°C for 5 minutes
• Hold at 4°C
Procedure
1
Place on ice and add 5 µl PPC.
2
Add 20 µl EPM, and then mix thoroughly as follows.
• [HS] Shake at 1600 rpm for 20 seconds.
• [LS] Pipette up and down.
3
Centrifuge at 280 × g for 1 minute.
4
Place on the thermal cycler and run the PCRNano program.
5
Centrifuge at 280 × g for 1 minute.
6
Add SPB. The volume depends on the type of adapter used.
Adapter Type
Adapter tubes
DAP
Volume SPB
50 µl
47.5 µl
7
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 (2–5 minutes).
11 Remove and discard all supernatant.
12 Wash 2 times with 200 µl 80% EtOH.
13 Use a 20 µl pipette to remove residual EtOH.
14 Air-dry on the magnetic stand for 5 minutes.
15 Add 32.5 µl RSB.
16 Remove from the magnetic stand, and then mix thoroughly as follows.
• [HS] Shake at 1800 rpm for 2 minutes.
• [LS] Pipette up and down.
17 Incubate at room temperature for 2 minutes.
10
Part # 15075697 Rev. A
Enrich DNA Fragments
18 [HS] Centrifuge at 280 × g for 1 minute.
19 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).
20 Transfer 30 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 Nano DNA Library Prep Protocol Guide
11
Validate Libraries
Procedure
12
1
Quantify the libraries using a fluorometric quantification method that uses dsDNA
binding dyes or qPCR.
2
Do the following on an Agilent Technologies 2100 Bioanalyzer:
• If using a High Sensitivity DNA chip:
— Dilute the DNA library 1:100 with water.
— Run 1 µl diluted DNA library.
• If using a DNA 7500 chip, run 1 µl undiluted DNA library.
Part # 15075697 Rev. A
Procedure
1
Transfer 10 µl library to the DCT plate.
2
Normalize with Tris-HCl 10 mM, pH 8.5 with 0.1% Tween 20 to 10 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 10 µl of each normalized library to a single well of
the PDP plate.
5
If pooling 25–48 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
If pooling 49–96 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 a 1.7 ml microcentrifuge tube.
7
Mix thoroughly as follows.
• [HS] Shake plate at 1800 rpm for 2 minutes or vortex the tube.
• [LS] Pipette up and down.
8
[HS] Centrifuge at 280 × g for 1 minute.
9
Proceed to cluster generation.
SAFE STOPPING POINT
If you are stopping, seal the plate or cap the tube and store at -25°C to -15°C for up to
7 days.
TruSeq Nano DNA Library Prep Protocol Guide
13
Normalize and Pool Libraries
Normalize and Pool Libraries
Acronyms
Acronym
14
Definition
ALP
Adapter Ligation Plate
ATL
A-Tailing Mix
CAP
Clean Up ALP Plate
CEP
Clean Up End Repair Plate
CSP
Clean Up Sheared DNA Plate
DAP
DNA Adapter Plate
DCT
Diluted Cluster Template Plate
DNA
Customer Sample DNA Plate
EPM
Enhanced PCR Mix
ERP
End Repair Mix
IMP
Insert Modification Plate
LIG
Ligation Mix
PDP
Pooled Dilution Plate
PPC
PCR Primer Cocktail
RSB
Resuspension Buffer
SPB
Sample Purification Beads
STL
Stop Ligation Buffer
TSP1
Target Sample Plate 1
Part # 15075697 Rev. A
For technical assistance, contact Illumina Technical Support.
Table 5 Illumina General Contact Information
Website
Email
www.illumina.com
[email protected]
Table 6 Illumina Customer Support Telephone Numbers
Region
Contact Number
Region
North America
1.800.809.4566
Italy
Australia
1.800.775.688
Netherlands
Austria
0800.296575
New Zealand
Belgium
0800.81102
Norway
Denmark
80882346
Spain
Finland
0800.918363
Sweden
France
0800.911850
Switzerland
Germany
0800.180.8994
United Kingdom
Ireland
1.800.812949
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 Nano DNA Library Prep Protocol Guide
Technical Assistance
Technical Assistance
*15075697*
Part # 15075697 Rev. A
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
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