TruSeq Synthetic Long-Read DNA Library Prep EUC and LTF (15047265 B)

TruSeq Synthetic Long-Read DNA Library Prep EUC and LTF (15047265 B)
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card and Lab Tracking Form
FOR RESEARCH USE ONLY
Date: __________________________
Illumina Kit Lot #: __________________________
Description: ______________________________________
_________________________________________________
NOTE
New or less experienced users are advised to follow the protocol in the TruSeq Synthetic Long-Read DNA Library
Prep Guide (part # 15047264) before using this document.
ILLUMINA PROPRIETARY
Catalog # FC-126-9002DOC
Part # 15047265 Rev. B
September 2014
Page 1 of 36
Page 2 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Consumables
Library Prep Kit Item
Lot Number
A-Tailing Mix (ATL)
Lot #: _____________________
End Repair Mix (ERP)
Lot #: _____________________
Ligation Mix (LIG)
Lot #: _____________________
Long Fragment Adapter (LAD)
Lot #: _____________________
MasterAmp Extra-Long DNA Polymerase Mix
Lot #: _____________________
qPCR Long-amp Primer Mix (QPM)
Lot #: _____________________
qPCR Master Mix (QMM)
Lot #: _____________________
qPCR Standard (QST)
Lot #: _____________________
Resuspension Buffer (RSB)
Lot #: _____________________
Sample Purification Beads (SPB)
Lot #: _____________________
Barcode Kit Item
Lot Number
Fragmentation Pre-Mix (FPM)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Gel Standard (GST)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Indexing Plate (IDP)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Long-amp Master Mix (LMM)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 3 of 36
Barcode Kit Item
Lot Number
Long-amp Primer Mix (LPM)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
MasterAmp Extra-Long DNA Polymerase Mix
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Resuspension Buffer (RSB)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Sample Neutralization Buffer (SNB)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Sample Purification Beads (SPB)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Tagment DNA Enzyme (TDE)
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Lot #: _____________________
Item
Date Prepared
80% Ethanol
Date Prepared: ____________
Page 4 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Fragment DNA
This process describes how to optimally fragment the gDNA using a g-TUBE for phasing and
long-read workflows.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
-25°C to -15°C
(2°C to 8°C after
initial thaw)
Illumina
gDNA samples
500 ng at 10 ng/µl per
sample
-25°C to -15°C
User
g-TUBE
1
15°C to 30°C
User
Microcentrifuge tubes
2
15°C to 30°C
User
Qubit dsDNA BR or HS assay kit
1
As specified by
manufacturer
User
Use IEM to create a sample sheet before beginning library preparation.
Sample Sheet Name: ________________________________________________
Procedure
[_] 1
Quantify the gDNA sample using the Qubit dsDNA BR or HS assay kit.
[_] 2
Normalize the gDNA sample with Resuspension Buffer to a final volume of 50 µl at
10 ng/µl in a new microcentrifuge tube.
[_] 3
Transfer 50 µl normalized gDNA to a g-TUBE.
[_] 4
Centrifuge the g-TUBE, with the blue cap up, to 4200 × g for 1 minute with a balance.
[_] 5
Flip the g-TUBE over, so that the blue cap is down, and centrifuge the tube one more time to
4200 × g for 1 minute with a balance.
[_] 6
Immediately remove the g-TUBE from the centrifuge.
[_] 7
Use a g-TUBE cap holder to transfer all of the fragmented DNA from the blue cap to the
microcentrifuge tube labeled gDNA-4200 × g along with the experiment date.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Perform End Repair on page 7, you can safely stop the
protocol here. If you are stopping, store the gDNA-4200 × g tube at 2°C to 8°C for up to 30 days.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 5 of 36
Page 6 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Perform End Repair
This process converts the overhangs resulting from fragmentation into blunt ends using End
Repair Mix. The 3' to 5' exonuclease activity of this mix removes the 3' overhangs and the 5' to
3' polymerase activity fills in the 5' overhangs.
Consumables
Item
Quantity
Storage
Supplied By
End Repair Mix (ERP)
1 tube per 4 reactions
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 4 reactions
2°C to 8°C
Illumina
Barcode labels for:
• LFP (Long Fragment Plate)
• LFP2 (Long Fragment Plate 2)
1 label per plate
15°C to 30°C
Illumina
96-well PCR plates
2
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Ice bucket
As needed
-25°C to -15°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Make LFP
[_] 1
Centrifuge the thawed End Repair Mix tube at 600 × g for 5 seconds.
[_] 2
Add 30 µl fragmented DNA sample from each gDNA-4200 × g tube to a separate well of the
new PCR plate labeled with the LFP barcode.
[_] 3
Add 20 µl End Repair Mix to each sample well of the plate. Set a 200 µl pipette to 40 µl,
and then gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 4
Seal the plate with a Microseal ‘B’ adhesive seal, then centrifuge the plate at 280 × g for
1 minute.
[_] 5
Return the End Repair Mix tube to -25°C to -15°C storage.
Incubate LFP
[_] 1
Place the sealed plate on the pre-programmed thermal cycler. Close the lid then select and
run the ERP program.
[_] a Choose the thermal cycler pre-heat lid option and set to 100°C
[_] b 30°C for 30 minutes
[_] c Hold at 4°C
[_] 2
Remove the plate from the thermal cycler when the program reaches 4°C.
[_] 3
Centrifuge the plate at 280 × g for 1 minute.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 7 of 36
Clean Up LFP
[_] 1
Remove the adhesive seal from the plate.
[_] 2
Vortex the Sample Purification Beads until they are well dispersed.
[_] 3
Add 80 µl well-mixed Sample Purification Beads to each well of the plate containing 50 µl
of the end repaired sample. Gently pipette the entire volume up and down 10 times to mix
thoroughly.
[_] 4
Incubate the plate at room temperature for 5 minutes.
Start time: _____________________
[_] 5
Stop time: _____________________
Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 6
Using a 200 µl single channel or multichannel pipette set to 127.5 µl, remove and discard
127.5 µl of supernatant from each well of the plate.
[_] 7
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each well
without disturbing the beads.
[_] 8
Incubate the plate at room temperature for 30 seconds, and then remove and discard all of
the supernatant from each well.
[_] 9
Repeat steps 7 and 8 one time for a total of two 80% EtOH washes.
[_] 10 Remove and discard any remaining EtOH from each well of the plate with a 10 µl pipette.
[_] 11 Let the plate stand at room temperature for 5 minutes to dry, and then remove the plate from
the magnetic stand.
Start time: _____________________
Stop time: _____________________
[_] 12 Add 20 µl Resuspension Buffer to each well of the plate. Gently pipette the entire volume up
and down 10 times to mix thoroughly.
Start time: _____________________
Stop time: _____________________
[_] 13 Incubate the plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 14 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 15 Transfer 17.5 µl of supernatant from each well of the LFP plate to the corresponding well of
the new PCR plate labeled with the LFP2 plate barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Adenylate 3' Ends on page 9, you can safely stop the
protocol here. If you are stopping, seal the LFP2 plate with a Microseal ‘B’ adhesive seal and store
at -25°C to -15°C for up to 7 days.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Page 8 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Adenylate 3' Ends
A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them from
ligating to one another during the adapter ligation reaction. A corresponding single
‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligating the
adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template)
formation.
Consumables
Item
Quantity
Storage
Supplied By
A-Tailing Mix (ATL)
1 tube per 4 reactions
-25°C to -15°C
Illumina
Ice bucket
As needed
-25°C to -15°C
User
Microseal ‘B’ adhesive seal
1
15° to 30°C
User
Add ATL
[_] 1
Centrifuge the thawed A-Tailing Mix tube at 600 × g for 5 seconds.
[_] 2
Add 12.5 µl thawed A-Tailing Mix to each well of the LFP2 plate. Set a 20 µl pipette to
20 µl, then gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 3
Seal the plate with a Microseal ‘B’ adhesive seal.
[_] 4
Return the A-Tailing Mix tube to -25°C to -15°C storage.
Incubate 1 LFP2
[_] 1
Centrifuge the plate at 280 × g for 1 minute.
[_] 2
Place the sealed plate, containing 30 µl of each sample, on the pre-programmed thermal
cycler. Close the lid, then select and run the ATAIL program.
[_] a Choose the pre-heat lid option and set to 100°C
[_] b 37°C for 30 minutes
[_] c Hold at 4°C
[_] 3
When the thermal cycler temperature is 4°C, remove the LFP2 plate from the thermal cycler,
then proceed immediately to Ligate Adapters on page 11.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 9 of 36
Page 10 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Ligate Adapters
This process ligates adapters to the ends of the long DNA fragments. These adapters are used as
markers in downstream data analysis processes, to denote the end of a contig.
Consumables
Item
Quantity
Storage
Supplied By
Ligation Mix (LIG)
1 tube per 4 reactions
-25°C to -15°C
Illumina
Long Fragment Adapters (LAD)
1 tube per 4 reactions
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 4 reactions
2°C to 8°C
Illumina
CLP (Cleaned Long Fragment
Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
96-well PCR plate
1
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Ice bucket
As needed
-25°C to -15°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Add LIG
[_] 1
Centrifuge the Long Fragment Adapters tube at 600 × g for 5 seconds.
[_] 2
Immediately before use, remove the Ligation Mix tube from -25°C to -15°C storage.
[_] 3
Centrifuge the LFP2 plate at 280 × g for 1 minute.
[_] 4
Remove the adhesive seal from the plate.
[_] 5
Add 5 µl Long Fragment Adapters to each sample well of the plate.
[_] 6
Add 2.5 µl Ligation Mix to each sample well of the plate. Set a 200 µl pipette to 30 µl, then
gently pipette the entire volume up and down 10 times to mix thoroughly.
[_] 7
Return the Ligation Mix tube back to -25°C to -15°C storage immediately after use.
[_] 8
Seal the plate with a Microseal ‘B’ adhesive seal, then centrifuge the plate at 280 × g for
1 minute.
Incubate 2 LFP2
[_] 1
Place the sealed plate, containing 37.5 µl of each sample, on the pre-programmed thermal
cycler. Close the lid then select and run the LIG program.
[_] a Choose the thermal cycler pre-heat lid option and set to 100°C
[_] b 30°C for 10 minutes
[_] c Hold at 4°C
[_] 2
Remove the plate from the thermal cycler when the program reaches 4°C.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 11 of 36
[_] 3
Centrifuge the plate at 280 × g for 1 minute.
Clean Up LFP2
[_] 1
Remove the adhesive seal from the plate.
[_] 2
Vortex the Sample Purification Beads for at least 1 minute or until they are well dispersed.
[_] 3
Add 37.5 µl well-mixed Sample Purification Beads to each sample well of the plate. Set a
200 µl pipette to 65 µl, and then gently pipette the entire volume up and down 10 times to
mix thoroughly.
[_] 4
Incubate the plate at room temperature for 5 minutes.
Start time: _____________________
[_] 5
Stop time: _____________________
Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 6
Remove and discard 70 µl of the supernatant from each well of the plate.
[_] 7
With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each sample
well without disturbing the beads.
[_] 8
Incubate the plate at room temperature for 30 seconds, and then remove and discard all of
the supernatant from each well.
[_] 9
Repeat steps 7 and 8 one time for a total of two 80% EtOH washes.
[_] 10 Remove and discard any remaining EtOH from each well of the plate with a 10 µl pipette.
[_] 11 With the plate on the magnetic stand, let the samples air-dry at room temperature for
5 minutes.
Start time: _____________________
Stop time: _____________________
[_] 12 With the plate on the magnetic stand, add 22.5 µl Resuspension Buffer to each sample well
of the plate.
[_] 13 Remove the plate from the magnetic stand.
[_] 14 Resuspend the beads in each well of the plate by repeatedly dispensing the Resuspension
Buffer over the bead pellet until it is immersed in the solution. Gently pipette the entire
volume up and down 10 times to mix thoroughly.
[_] 15 Incubate the plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 16 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 17 Transfer 20 µl of the supernatant from each well of the LFP2 plate to the corresponding well
of the new PCR plate labeled with the CLP barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Purify Ligation Products and Size Selection on page 13,
you can safely stop the protocol here. If you are stopping, seal the CLP plate with a Microseal ‘B’
adhesive seal and store at 2°C to 8°C overnight.
Page 12 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Purify Ligation Products and Size Selection
This process purifies the products of the ligation reaction on a gel and removes unligated
adapters, as well as any adapters that might have ligated to one another. Long adapter ligated
fragments of DNA of 8–10 kb in size are selected for Long Range PCR and subsequent
Tagmentation procedures.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
1 Kb DNA Extension Ladder
0.5 µl per sample
15°C to 30°C
User
2-propanol (Isopropanol)
1 µl × the mg weight
of each gel slice
15°C to 30°C
User
E-Gel NGS, 0.8% Agarose
1 per sample
15°C to 30°C
User
Fragmented gDNA-4200 × g
5 µl per sample
15°C to 30°C
User
Lab pen
1
15C° to 30°C
User
Microcentrifuge tubes
2 per sample + 2
15°C to 30°C
User
QIAquick Gel Extraction Kit
1
15°C to 30°C
User
Ruler
1
15C° to 30°C
User
X-tracta Gel Extraction Tool
1 per sample
15C° to 30°C
User
Size Separate
[_] 1
Place one E-Gel NGS, 0.8% Agarose per sample into an E-Gel iBase Power System according
to manufacturer instructions.
[_] 2
Add 0.5 µl 1 Kb DNA Extension Ladder to 19.5 µl Resuspension Buffer in a microcentrifuge
tube to dilute the DNA ladder. Multiply each reagent volume by the number of gels being
prepared. Gently pipette the entire volume up and down 6-8 times to mix thoroughly.
[_] 3
Add 5 µl of 10 ng/µl fragmented DNA sample from the tube labeled gDNA-4200 × g to 15 µl
Resuspension Buffer in a microcentrifuge tube to dilute the fragmented gDNA. Multiply each
reagent volume by the number of gels being prepared. Gently pipette the entire volume up
and down 6–8 times to mix thoroughly.
[_] 4
Centrifuge the diluted fragmented gDNA tube at 280 × g for 1 minute.
[_] 5
Load 20 µl sample from one well of the CLP plate into lane 4 of one gel.
[_] 6
Repeat step 5 for each sample, loading a single sample into lane 4 of each gel.
[_] 7
Load 20 µl diluted 1 Kb DNA Extension Ladder into lane 6 of each gel.
[_] 8
Load 20 µl diluted fragmented gDNA sample into lane 8 of each gel.
[_] 9
Load each empty well of each gel with 20 µl Resuspension Buffer.
[_] 10 On the E-Gel iBase Power System, select and run the E-Gel 0.8-2% program. Set the run time
to 26 minutes.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 13 of 36
[_] 11 View the gel on a Dark Reader transilluminator.
[_] 12 Use a lab pen and ruler to mark the 8–10 kb region of interest for precise gel excision, as
follows:
[_] a Draw two vertical lines on the plastic gel cassette to mark the left and right side of the
sample well.
[_] b Draw two horizontal lines on the plastic gel cassette to mark the position of the 10 kb
and 8 kb bands of the ladder.
[_] c Repeat step a and b on the other side of plastic cassette, because the gel can stick to
either side of cassette when it is opened.
Purify Gel
[_] 1
Carefully open the gel with the Novex Gel Knife.
[_] 2
View the gel on a Dark Reader transilluminator to confirm the correct placement of the lines
drawn on the plastic gel cassette at the 8-10 kb region of interest. Carefully adjust the
position of the gel if it has shifted relative to the marked region.
[_] 3
Place an x-tracta tool on the gel between the marked region of interest on the plastic gel
cassette and press the tool into the gel.
[_] 4
Rock the x-tracta tool side to side to extract the desired gel slice.
[_] 5
Place the x-tracta tool over the appropriately labeled microcentrifuge tube and expel the
extracted gel band from the tool into the tube with a quick squeeze.
[_] 6
Weigh the tube containing the gel slice. Subtract the weight of the empty tube to determine
weight of the gel slice in milligrams (mg).
Sample
1
2
3
4
Tube Weight
Empty Tube Weight
Gel Slice Weight (mg)
[_] 7
For each sample, add X µl QIAGEN Buffer QG, with X equaling three times the mg weight of
the gel slice, to the tube containing the gel slice.
[_] 8
Place the tube containing the gel and QIAGEN Buffer QG mixture on the pre-heated
microheating system or water bath. Close the lid and incubate at 50°C for 10 minutes to melt
the gel. Gently flick the tube periodically until the gel is fully melted.
Start time: _____________________
[_] 9
Stop time: _____________________
Add 1 µl Isopropanol × the mg weight of the gel slice to the gel and QIAGEN Buffer QG
mixture.
[_] 10 Add the dissolved gel, QIAGEN Buffer QG, and Isopropanol mixture to a QIAquick column.
[_] 11 Centrifuge the QIAquick column to 13,000 rpm for 1 minute.
[_] 12 Remove and discard the eluate from the QIAquick column.
[_] 13 Add 750 µl PE buffer (with ethanol added) to the QIAquick column.
[_] 14 Centrifuge the QIAquick column to 13,000 rpm for 1 minute.
[_] 15 Remove and discard the supernatant from the QIAquick column.
[_] 16 Centrifuge the QIAquick column to 13,000 rpm for 1 minute.
Page 14 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
[_] 17 Remove and discard the supernatant from the QIAquick column.
[_] 18 Remove the QIAquick column from the collection tube and place it in the new
microcentrifuge tube labeled size-selected [sample name].
[_] 19 Add 52 µl Resuspension Buffer to the QIAquick column in the microcentrifuge tube.
[_] 20 Incubate the microcentrifuge tube at room temperature for 1 minute.
[_] 21 Centrifuge the QIAquick column in the microcentrifuge tube at 13,000 rpm for 1 minute.
[_] 22 Discard the QIAquick column.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Validate Library on page 17, you can safely stop the
protocol here. If you are stopping, cap the size-selected [sample name] tube and store at
2°C to 8°C for up to 3 months. Avoid a freeze-thaw cycle.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 15 of 36
Page 16 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Validate Library
Perform the following procedures for quality control analysis and quantification of the long DNA
fragments.
Quantify Libraries
Quantify 2 µl of the library using the Qubit dsDNA HS Assay Kit. The library should yield
> 0.05 ng/µl.
[Optional] Quality Control
To verify the size of your fragments, check the template size distribution.
Run 1 µl of the DNA library on an Agilent Technologies 2100 Bioanalyzer using a High
Sensitivity DNA chip. The peak partially overlaps with 10 kb upper marker.
Comments
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__________________________________________________________________________________
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 17 of 36
Page 18 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
qPCR Quantitation
This process quantifies the long DNA fragments to make sure that the appropriate amount of
DNA is used for the Long Range PCR and subsequent Tagmentation procedures.
Consumables
Item
Quantity
Storage
Supplied By
MasterAmp Extra-Long DNA
Polymerase Mix
0.4 µl per reaction
-25°C to -15°C
Illumina
qPCR Long-amp Primer Mix
(QPM)
2 µl per reaction
-25°C to -15°C
Illumina
qPCR Master Mix (QMM)
11.6 µl per reaction
-25°C to -15°C
Illumina
qPCR Standard (QST)
5 µl per standard
curve
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
QLP (Quantification Long
Fragment Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
Dimethyl sulfoxide (DMSO)
1 ml
15°C to 30°C
User
Ice bucket
As needed
-25°C to -15°C
User
PCR-grade water
1 ml
15°C to 30°C
User
RNase/DNase-free eight-tube
strip with caps
1
15°C to 30°C
User
Microcentrifuge tubes
1 per sample + 3
15°C to 30°C
User
qPCR plate and seal
1
15°C to 30°C
User
ROX Reference Dye 50x
As specified by
manufacturer
-25°C to -15°C
User
SYBR Green 10,000x
5 µl
-25°C to -15°C
User
Prepare SYBR Green
[_] 1
Vortex the thawed SYBR Green 10,000x to mix thoroughly.
[_] 2
Add 5 µl SYBR Green 10,000x and 495 µl of DMSO to a microcentrifuge tube to dilute the
SYBR Green to 100x. Vortex the solution to mix thoroughly.
[_] 3
Measure the absorbance of 100x diluted SYBR Green on a NanoDrop instrument. The ideal
Abs494±3 nm of 100x SYBR Green stock is 0.5–0.6, which indicates that the concentration is
100x. Adjust the concentration if necessary. For more information, see Calibrate Diluted SYBR
Green in the TruSeq Synthetic Long-Read DNA Library Prep Guide (part # 15047264).
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 19 of 36
Dilute qPCR Standard
[_] 1
Using a 200 µl single channel pipette set to 20 µl, gently pipette the qPCR Standard up and
down 10 times to mix thoroughly, then centrifuge briefly.
[_] 2
Add Resuspension Buffer to the labeled tubes as follows:
Tube Label
Tube Type
QST 1:100 Dilution
Std2
Std3
Std4
NTC (no template control)
[Sample name] 1:100 Dilution
Microcentrifuge
Eight-tube strip
Eight-tube strip
Eight-tube strip
Eight-tube strip
Microcentrifuge
Resuspension Buffer
Volume (μl)
495
45
45
45
50
495
[_] 3
Add 5 µl qPCR Standard to the QST 1:100 Dilution tube for a total of 10 pg/µl (10,000 fg/µl).
Using a 1000 µl single channel or multichannel pipette, gently pipette the entire volume up
and down 6–8 times to mix thoroughly.
[_] 4
Centrifuge the QST 1:100 Dilution tube at 600 × g for 5 seconds.
[_] 5
Transfer 50 µl from the QST 1:100 Dilution tube to the Std 1 tube in the eight-tube strip.
Change the tip.
[_] 6
Transfer 5 µl from the Std1 tube to the Std2 tube for a total of 1 pg/µl (1000 fg/µl). Using a
200 µl single channel pipette set to 45 µl, gently pipette the entire volume up and down 6–
8 times to mix thoroughly. Change the tip.
[_] 7
Transfer 5 µl from the Std2 tube to the Std3 tube for a total for a total of 0.1 pg/µl (100 fg/µl).
Using a 200 µl single channel pipette set to 45 µl, gently pipette the entire volume up and
down 6–8 times to mix thoroughly. Change the tip.
[_] 8
Transfer 5 µl from the Std3 tube to the Std4 tube for a total of 0.01 pg/µl (10 fg/µl). Using a
200 µl single channel pipette set to 45 µl, gently pipette the entire volume up and down 6–
8 times to mix thoroughly. Discard the tip.
[_] 9
Cap the eight-tube strip that contains the serial diluted qPCR Standard, then centrifuge
briefly. This serves as the qPCR standard in the Long Range PCR procedure.
Dilute Sample
[_] 1
Add 5 µl size-selected DNA from the microcentrifuge tube from step 21 of Purify Gel on page
14 to each tube labeled with the [sample name] 1:100 Dilution. Using a 1000 µl single
channel or multichannel pipette, gently pipette the entire volume up and down 6–8 times to
mix thoroughly.
[_] 2
Cap and store the size-selected [sample name] tubes at 2°C to 8°C for up to 90 days.
Prepare Master Mix
[_] 1
Prepare a fresh dilution of 1.5x SYBR Green from 100x SYBR Green stock (3 µl 100x SYBR
Green in 197 µl PCR-grade water) to create a dye mix. If ROX is required for your qPCR
instrument, dilute SYBR Green and ROX dye together to make a 1.5x SYBR Green/10x ROX
dye mixture.
Page 20 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
[_] 2
Set up a master mix in a sterile, nuclease-free microcentrifuge tube on ice using the
following. Using a 1000 µl single channel or multichannel pipette, gently pipette the entire
volume up and down 6–8 times to mix thoroughly.
Reagent
qPCR Master Mix
qPCR Long-amp Primer Mix
Dye Mix
(1.5x SYBR Green with optional 10x ROX)
MasterAmp Extra-long DNA Polymerase
Mix
Total volume
1 Sample
2 Samples
3 Samples
4 Samples
255 µl
44 µl
44 µl
302 µl
52 µl
52 µl
336 µl
58 µl
58 µl
394 µl
68 µl
68 µl
9 µl
10.5 µl
11.5 µl
13.5 µl
352 μl
416.5 μl
463.5 μl
543.5 μl
[_] 3
Add 16 µl master mix to each required well of the plate labeled with the QLP barcode.
[_] 4
Remove the cap from the standard eight-strip tube from step 9 of Dilute qPCR Standard on
page 20.
[_] 5
Add 4 µl Std 1 to each well in rows C–E, column 4.
[_] 6
Add 4 µl Std 2 to each well in rows C–E, column 5. Change the tip.
[_] 7
Add 4 µl Std 3 to each well in rows C–E, column 6 Change the tip.
[_] 8
Add 4 µl Std 4 to each well in rows C–E, column 7. Change the tip.
[_] 9
Add 4 µl NTC to each well in rows C–E, column 8. Change the tip.
[_] 10 Remove the cap from the microcentrifuge tube that contains each one 1:100 Dilution sample.
[_] 11 Add 4 µl of one 1:100 Dilution sample to each well in rows C–E, column 9.
[_] 12 Add 4 µl of each additional 1:100 Dilution sample to each well in rows C–E, adding one
column per sample.
[_] 13 Cap and store the [sample name] 1:100 Dilution tubes at 2°C to 8°C for subsequent use in
this protocol. The samples can be stored for up to 30 days.
[_] 14 Mix the plate thoroughly as follows:
[_] a Seal the plate with an appropriate adhesive seal for the plate.
[_] b Shake the plate on a microplate shaker at 1600 rpm for 30 seconds.
[_] 15 Centrifuge the plate at 280 × g for 1 minute.
[_] 16 Place the sealed plate on the qPCR instrument. Close the lid then and run the instrument as
follows:
[_] a 94°C for 1 minute
[_] b 40 cycles of:
— 94°C for 30 seconds
— 65°C for 30 seconds
— 68°C for 10 minutes
[_] c [Optional] Melting Curve setting suggested by qPCR instrument
[_] 17 Remove the plate from the qPCR instrument.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 21 of 36
Analysis
Do one of the following:
[_] 1 If you are using qPCR instrument software to annotate standards and sample concentration:
[_] a Calculate the average Cq value of the qPCR standards and 1:100 dilution of sample from
triplicate wells in the QLP plate.
[_] b Use the qPCR instrument software to annotate standards as follows:
Std1
Std2
Std3
Std4
[_] c
[_] d
[_] e
Concentration (pg/μl)
10
1
0.1
0.01
Confirm that the qPCR reaction efficiency is 50–100%, which is a typical reaction
efficiency of a long qPCR amplicon.
Confirm that the R2 of the best fit line is > 0.97. Poor R2 values can indicate a dilution
error in the standard curve or poor amplification of one or more of the standards.
Use the average of the triplicate data points corresponding 1:100 sample dilution to
calculate the concentration of the sample.
[_] 2 If you are using a graphing program to manually calculate sample concentration:
[_] a Calculate the average Cq value of the qPCR standards and 1:100 dilution of sample from
triplicate wells in the QLP plate.
[_] b Create a scatter plot of the average Cq of the qPCR standards on the X-axis and the log
base 2 value of the DNA concentration (pg/µl) of the qPCR standards on the Y-axis.
[_] c Determine the equation of the best fit line for the qPCR standard curve values, which is
in the format of y = mx + b. This is equivalent to: log base 2 DNA concentration = (slope
× Cq) + y_int.
[_] d Confirm that the qPCR reaction efficiency (the slope in the equation in step c) is 50-100%,
which is a typical reaction efficiency of a long qPCR amplicon.
[_] e Confirm that the R2 of the best fit line is > 0.97. Poor R2 values can indicate a dilution
error in the standard curve or poor amplification of one or more of the standards.
[_] f
Determine the value of y in y = mx + b by using the average Cq of each 1:100 dilution of
sample for x in the equation.
[_] g Calculate the concentration of each 1:100 dilution of sample in pg/µl, using the
following equation, where Concentration (pg/µl) = 2 ^ y:
Sample average Cq = 14.6
y = (-0.935 × 14.6) + 12.431 = -1.226
Concentration of 1:100 dilution of sample = 2 ^ -1.226 = 0.428 pg/µl
Comments
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Page 22 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Long Range PCR
This process enriches long DNA fragments with the appropriate adapters. The PCR starting
material is diluted in a 384-well plate to limit the number of molecules in each well, which
enables downstream data-analysis applications. The PCR-amplified material is subject to gel
quality control to make sure that the material is not over- or under-amplified.
Consumables
Item
Quantity
Storage
Supplied By
Gel Standard (GST)
1 tube
-25°C to -15°C
Illumina
Long-amp Master Mix (LMM)
1 tube
-25°C to -15°C
Illumina
Long-amp Primer Mix (LPM)
1 tube
-25°C to -15°C
Illumina
MasterAmp Extra-Long DNA
Polymerase Mix
1 tube
-25°C to -15°C
Illumina
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
LAP (Long Fragment
Amplification Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
E-Gel EX Agarose Gel, 1%
1
15°C to 30°C
User
2-Log DNA Ladder
1
15°C to 30°C
User
15 ml conical tube
1
15°C to 30°C
User
96-well PCR plate or
RNase/DNase-free eight-tube
strip with caps
1
15°C to 30°C
User
384-well PCR plate
1
15°C to 30°C
User
Ice bucket
As needed
-25°C to -15°C
User
Microcentrifuge tubes
6
15°C to 30°C
User
Microseal ‘B’ adhesive seals
4
15°C to 30°C
User
Needle (22 1/2 gauge)
1 per sample
15°C to 30°C
User
RNase/DNase-free eight-tube
strips with caps
2
15°C to 30°C
User
RNase/DNase-free reagent
reservoir
1
15°C to 30°C
User
Dilute Template
[_] 1
Determine if the 1:100 diluted library template is of sufficient quantity for Long Range PCR.
• For the Phasing workflow, 75 fg library is required per well. A total of 37,500 fg library
per plate.
• For the Long-Read workflow, 3 fg library is required per well. A total of 1500 fg library
per plate.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 23 of 36
[_] 2
If there is not enough library template to make the dilution, use the undiluted template from
step 2 of Dilute Sample on page 20.
[_] 3
Dilute the library template with Resuspension Buffer to the following concentration with a
total volume of 750 µl.
• For the Phasing workflow, dilute the library template to 50 fg/µl.
• For the Long-Read workflow, dilute the library template to 2 fg/µl.
Sample
1:100 Diluted Library
(fg/μl)
Diluted Library (μl)
Resuspension Buffer
(μl)
Total Volume
(μl)
1
2
3
4
Prepare PCR Master Mix
[_] 1
Set up a PCR master mix in a sterile, nuclease-free 15 ml conical tube on ice using the
following:
Reagent
Diluted template
Long-amp Master Mix
Long-amp Primer Mix
MasterAmp Extra-long DNA Polymerase Mix
Total Volume
Volume (μl)
750
1450
250
50
2500
[_] 2
Cap the tube and gently invert the tube several times to mix.
[_] 3
Aliquot 280 µl PCR master mix into each well of an eight-tube strip.
[_] 4
Set a 200 µl electronic eight-channel pipette to 120 µl and 5 µl per dispense, for a total of
24 dispenses.
[_] 5
Add 5 µl PCR master mix to each well of the new 384-well PCR plate labeled with the LAP
barcode.
[_] 6
Repeat steps 4 and 5 one time.
[_] 7
Quickly seal the plate with a Microseal ‘B’ adhesive seal, then centrifuge the plate at 500 × g
for 1 minute.
[_] 8
Cap the PCR master mix eight-tube strip and keep the strip on ice
Long Amp Plate
[_] 1
Place the sealed plate on the 384-well thermal cycler and place a compression mat on top of
the plate. Close the lid then select and run the Phasing15 or LongRead21 program,
depending on the workflow.
[_] 2
While the thermal cycler is running, proceed to Long Amp Quality Control.
Long Amp Quality Control
[_] 1
Add 50 µl PCR master mix to one well of a new PCR plate or an eight-tube strip.
[_] 2
Seal the plate with a Microseal ‘B’ adhesive seal or cap the eight-tube strip.
Page 24 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
[_] 3
Place the sealed plate or capped tube on the 96-well thermal cycler. Close the lid then
select and run the Phasing20QC or LongRead26QC program, depending on the workflow.
[_] 4
Remove the LAP plate and 96-well PCR plate or eight-tube strip from both thermal cyclers
and place them on ice.
Gel Quality Control
[_] 1
Add Resuspension Buffer to the labeled microcentrifuge tubes as follows:
Tube
GST1
GST2
GST3
GST4
Resuspension Buffer
Volume (μl)
36
20
20
20
[_] 2
Add 4 µl undiluted Gel Standard to the GST1 tube for a total of 0.1 ng/µl. Gently pipette the
entire volume up and down 6–8 times to mix thoroughly. Change the tip.
[_] 3
Transfer 20 µl from the GST1 tube to the GST2 tube for a total of 0.05 ng/µl. Gently pipette
the entire volume up and down 6–8 times to mix thoroughly. Change the tip.
[_] 4
Transfer 20 µl from the GST2 tube to the GST3 tube for a total of 0.025 ng/µl. Gently pipette
the entire volume up and down 6–8 times to mix thoroughly. Change the tip.
[_] 5
Transfer 20 µl from the GST3 tube to the GST4 tube for a total of 0.0125 ng/µl. Gently
pipette the entire volume up and down 6–8 times to mix thoroughly.
[_] 6
Centrifuge the LAP plate at 500 × g for 1 minute.
[_] 7
Using 22 1/2 gauge needle, carefully pierce a hole in the plate seal above four randomly
selected wells of the plate.
[_] 8
Transfer 5 µl from each of four randomly selected plate wells to pool in one well of an eighttube strip. Note which wells were selected from the plate.
Sample
1
2
3
4
[_] 9
LAP Plate Well
Place the LAP plate on ice or store the plate at 2°C to 8°C for up to 24 hours or until this
Long Range PCR procedure is complete.
[_] 10 Cap the eight-tube strip that contains the pooled samples, briefly centrifuge the strip at
500 × g.
[_] 11 Add 0.5 µl 2-Log DNA Ladder and 19.5 µl Resuspension Buffer to the tube labeled 2-Log
Ladder to dilute. Gently pipette the entire volume up and down 6–8 times to mix
thoroughly.
[_] 12 Load all of the Diluted 2-Log DNA Ladder into the well of lane 1 of a E-Gel EX 1%.
[_] 13 Load 20 µl from the GST1 tube into the well of lane 2 of the gel.
[_] 14 Load 20 µl from the GST2 tube into the well of lane 3 of the gel.
[_] 15 Load 20 µl from the GST3 tube into the well of lane 4 of the gel.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 25 of 36
[_] 16 Load 20 µl from the GST4 tube into the well of lane 5 of the gel.
[_] 17 Load 20 µl pooled sample into the well of lane 6 of the gel.
[_] 18 Load 20 µl quality control sample, from the conclusion of Long Amp Quality Control on page
24, into the well of lane 7 of the gel.
[_] 19 Load each of the empty wells with 20 µl Resuspension Buffer.
[_] 20 Select and run the E-Gel EX 1–2% program.
[_] 21 View the gel on a Dark Reader transilluminator.
The pooled sample and QC sample bands should migrate the same distance and the pooled
sample intensity should be between the intensity of GST1 (0.1 ng/µl) and GST4 (0.0125 ng/µl). The Gel Standard migrates in the gel as a single band at 10 kb.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Tagmentation on page 27, you can safely stop the
protocol here. If you are stopping, seal the LAP plate with a Microseal ‘B’ adhesive seal and store at
2°C to 8°C for up to 24 hours.
Comments
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Page 26 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Tagmentation
This process tagments (tags and fragments) PCR amplified long DNA fragments by adding the
Nextera transposome to the 384-well plate. The Nextera transposome simultaneously fragments
the genomic DNA and adds adapter sequences to the ends, allowing for amplification by PCR in
subsequent procedures.
Consumables
Item
Quantity
Storage
Supplied By
Fragmentation plate
1
15°C to 30°C
Illumina
Fragmentation Pre-Mix (FPM)
1 tube per LAP plate
-25°C to -15°C
Illumina
Tagment DNA Enzyme (TDE)
1 tube per LAP plate
-25°C to -15°C
Illumina
Ice bucket
As needed
-25°C to -15°C
User
Microcentrifuge tube
1
15°C to 30°C
User
Microseal ‘B’ adhesive seals
2
15°C to 30°C
User
Procedure
[_] 1
Centrifuge the LAP plate at 500 × g for 1 minute.
[_] 2
Remove the seal from the LAP plate, then place the Fragmentation plate on top of LAP plate.
[_] 3
Add 36 µl Tagment DNA Enzyme and 1464 µl Fragmentation Pre-Mix to a microcentrifuge
tube.
[_] 4
Invert the tube 10 times to mix thoroughly, then centrifuge briefly.
[_] 5
Transfer 180 µl of the mixture to each well of an eight-tube strip.
[_] 6
Set a 200 µl electronic eight-channel pipette to 144 µl and 3 µl per dispense.
[_] 7
Add 3 µl Tagment DNA Enzyme and Fragmentation Pre-Mix to each well of the
Fragmentation plate that is on top of the LAP plate.
[_] 8
Centrifuge the stacked Fragmentation plate and LAP plates to 500 × g for 1 minute.
[_] 9
Place the stacked Fragmentation plate and LAP plates on the benchtop, with the LAP plate
on the bottom.
[_] 10 Carefully remove the Fragmentation plate from the LAP plate and discard the Fragmentation
plate.
[_] 11 Mix the LAP plate thoroughly as follows:
[_] a Seal the plate with a Microseal ‘B’ adhesive seal.
[_] b Shake the plate on a microplate shaker at 1600 rpm for 30 seconds.
[_] 12 Centrifuge the plate at 500 × g for 1 minute.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 27 of 36
[_] 13 Place the sealed plate on the thermal cycler and place a compression mat on top of the plate.
Close the lid and then select and run the Tag program.
[_] a Choose the thermal cycler pre-heat lid option and set to 100°C
[_] b 55°C for 15 minutes
[_] c 4°C for 5 minutes
[_] d 72°C for 4 minutes
[_] e Hold at 4°C
[_] 14 Remove the LAP plate from the thermal cycler.
Comments
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Page 28 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Indexing PCR
This process amplifies tagmented DNA by PCR. A unique index and the P5 and P7 adapters are
added to the tagmented DNA in each well of the 384-well plate. The P5 and P7 adapters are
required for cluster generation and sequencing.
Consumables
Item
Quantity
Storage
Supplied By
Indexing Plate (IDP)
1
-25°C to -15°C
Illumina
[Optional] Alignment
ring
1 per LAP plate
15°C to 30°C
Illumina
Microseal ‘B’ adhesive
seals
3
15°C to 30°C
User
Procedure
[_] 1
Centrifuge the LAP plate at 500 × g for 1 minute.
[_] 2
Centrifuge the IDP plate at 500 × g for 1 minute.
[_] 3
Make sure that the droplets are at the bottom of each well of the IDP plate.
[_] 4
Remove the adhesive seal from the LAP plate.
[_] 5
Remove the foil seal from the IDP plate.
[_] 6
[Optional] Place the alignment ring on the LAP plate so that the notched corners align.
[_] 7
Invert the IDP plate.
[_] 8
Carefully place the inverted IDP plate on top of the LAP plate, so that the corner notches and
wells of both plates align.
[_] 9
Centrifuge the stacked IDP and LAP plates to 500 × g for 1 minute. Make sure that the LAP
plate is on the bottom.
[_] 10 Place the stacked IDP and LAP plates on the benchtop, with the LAP plate on the bottom.
[_] 11 Carefully remove the IDP plate from the LAP plate and place it, the top side facing up, on
the benchtop.
[_] 12 Mix the LAP plate thoroughly as follows:
[_] a Seal the plate with a Microseal ‘B’ adhesive seal.
[_] b Shake the plate on a microplate shaker at 1600 rpm for 30 seconds.
[_] 13 Centrifuge the plate at 500 × g for 1 minute.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 29 of 36
[_] 14 Place the sealed plate on the thermal cycler and place a compression mat on top of the plate.
Close the lid then select and run the PostTagAmp program.
[_] a Choose the thermal cycler pre-heat lid option and set to 100°C
[_] b 94°C for 1 minute
[_] c 10 cycles of:
— 94°C for 15 seconds
— 65°C for 4 minutes
[_] d Hold at 4°C for up to one hour
[_] 15 Remove the LAP plate from the thermal cycler.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Pool and Concentrate on page 31, you can safely stop
the protocol here. If you are stopping, store the LAP plate at -25°C to -15°C for up to 7 days or at
2°C to 8°C for up to 24 hours.
Comments
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Page 30 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Pool and Concentrate
This process collects and concentrates the PCR-amplified and indexed DNA from the 384-well
plate into a single sample.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Neutralization Buffer
(SNB)
1 tube per 1 reaction
2°C to 8°C
Illumina
Collection plate
1 per sample
15°C to 30°C
Illumina
PAP (Pooled
Amplicon Plate) barcode label
1 label per plate
15°C to 30°C
Illumina
50 ml conical tube
1 per sample
15°C to 30°C
User
96-well MIDI plate
1
15°C to 30°C
User
Microcentrifuge tube
2 per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seal
1
15°C to 30°C
User
RNase/DNase-free eight-tube
strip and caps
1 per sample
15°C to 30°C
User
Zymo DNA Binding Buffer
1
15°C to 30°C
User
Zymo DNA Wash Buffer (with
ethanol added)
1
15°C to 30°C
User
Zymo-Spin V Column with
Reservoir
1
15°C to 30°C
User
Procedure
[_] 1
Centrifuge the LAP plate at 500 × g for 1 minute.
[_] 2
Remove the adhesive seal from the LAP plate, then attach the collection plate to the LAP
plate, so that the LAP plate is covered with the collection plate.
[_] 3
Invert the attached collection and LAP plates so that the sample plate wells face down into
the collection plate.
[_] 4
Centrifuge the collection and LAP plates with a balance to 500 × g for 30 seconds.
[_] 5
Set up a master mix in a new, sterile, nuclease-free 50 ml conical tube using the following:
Reagent
All of the pooled library from the collection plate
Sample Neutralization Buffer
Zymo DNA Binding Buffer
Total Volume
[_] 6
Volume
4–5 ml
200 µl
20 ml
~24.5 ml
Cap the master mix tube and invert the tube several times to mix.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 31 of 36
[_] 7
Set up a Zymo-Spin V column with reservoir on a vacuum manifold.
[_] 8
Turn on the vacuum and leave it on.
[_] 9
Add 12 ml master mix to the Zymo-Spin V column.
[_] 10 Run the master mix through the vacuum until all of the liquid has passed through the
Zymo-Spin V column and into the vacuum manifold.
[_] 11 Add the remaining master mix to the Zymo-Spin V column.
[_] 12 Run the master mix through the vacuum until all of the liquid has passed through the
Zymo-Spin V column and into the vacuum manifold.
[_] 13 Add 4 ml Zymo DNA Wash Buffer (with ethanol added) to the Zymo-Spin V column to
wash the sample while it is on the vacuum.
[_] 14 Remove the Zymo-Spin V column from the vacuum manifold and unattach the reagent
reservoir from the column.
[_] 15 Discard reagent reservoir
[_] 16 Centrifuge Zymo-Spin V column at 11,000 × g for 1 minute in a microcentrifuge tube to
remove any residual Zymo DNA Wash Buffer.
[_] 17 Place the Zymo-Spin V column into a new microcentrifuge tube, then add 160 µl
Resuspension Buffer to the column.
[_] 18 Centrifuge the microcentrifuge tube at 10,000 × g for 1 minute to collect the eluate.
[_] 19 Transfer 150 µl from the microcentrifuge tube to a single well of the new MIDI plate labeled
with the PAP barcode.
[_] 20 Transfer 5 µl from the microcentrifuge tube to the new eight-tube strip labeled QC1: Pre Size
Selection.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Size Selection on page 33, you can safely stop the
protocol here. If you are stopping, seal the PAP plate with a Microseal ‘B’ adhesive seal. Store the
plate at -25°C to -15°C for up to 7 days or at 2°C to 8°C for up to 24 hours.
Comments
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Page 32 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Size Selection
This process removes adapter dimers and DNA fragments that are either too small or too large,
selecting for tagmented DNA in the optimal range for cluster formation.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
Sample Purification Beads (SPB)
1 tube per 24 reactions
2°C to 8°C
Illumina
FSP (Final Sample Plate) barcode
label
1 label per plate
15°C to 30°C
Illumina
96-well MIDI plate
1
15°C to 30°C
User
Freshly prepared 80% ethanol
(EtOH)
400 µl per sample
15°C to 30°C
User
Microseal ‘B’ adhesive seals
3
15°C to 30°C
User
Procedure
[_] 1
Vortex the Sample Purification Beads until they are well dispersed.
[_] 2
Add 67.5 µl well-mixed Sample Purification Beads to each sample well of the PAP plate.
Mix thoroughly as follows:
[_] a Seal the plate with a Microseal ‘B’ adhesive seal.
[_] b Shake the plate on a microplate shaker at 1600 rpm for 2 minutes or until the beads are
well dispersed.
[_] 3
Incubate the plate at room temperature for 5 minutes.
Start time: _____________________
Stop time: _____________________
[_] 4
Centrifuge the plate at 280 × g for 1 minute.
[_] 5
Remove the adhesive seal from the plate, then place the plate on the magnetic stand for
5 minutes or until the liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 6
Using a 200 µl single channel or multichannel pipette set to 106 µl, transfer 106 µl of the
supernatant, containing the DNA of interest, from each sample well of the plate to an empty
well in the same plate.
[_] 7
Repeat step 6 one time, transferring each sample to the same well that the sample was
transferred to in step 6. Each plate sample well now contains a total of 212 μl of DNA of
interest.
[_] 8
Remove the plate from the magnetic stand.
[_] 9
Add 30 µl well-mixed Sample Purification Beads to each well of the plate. Mix thoroughly
as follows:
[_] a Seal the plate with a Microseal ‘B’ adhesive seal.
[_] b Shake the plate on a microplate shaker at 1600 rpm for 2 minutes.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 33 of 36
[_] 10 Incubate the plate at room temperature for 5 minutes.
Start time: _____________________
Stop time: _____________________
[_] 11 Centrifuge the plate at 280 × g for 1 minute.
[_] 12 Remove the adhesive seal from the plate.
[_] 13 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 14 Remove and discard all of the supernatant from each well of the plate.
[_] 15 With the plate on the magnetic stand, add 200 µl freshly prepared 80% EtOH to each well
with a sample without disturbing the beads.
[_] 16 Incubate the plate at room temperature for 30 seconds, and then remove and discard all of
the supernatant from each well.
[_] 17 Repeat steps 15 and 16 one time for a total of two 80% EtOH washes.
[_] 18 Remove and discard any remaining EtOH from each well of the plate with a 10 µl pipette.
[_] 19 Let the plate stand at room temperature for 5 minutes to dry, and then remove the plate from
the magnetic stand.
Start time: _____________________
Stop time: _____________________
[_] 20 Resuspend the dried pellet in each well with 32.5 µl Resuspension Buffer. Gently pipette the
entire volume up and down 10 times to mix thoroughly.
Start time: _____________________
Stop time: _____________________
[_] 21 Incubate the plate at room temperature for 2 minutes.
Start time: _____________________
Stop time: _____________________
[_] 22 Place the plate on the magnetic stand for 5 minutes or until the liquid is clear.
Start time: _____________________
Stop time: _____________________
[_] 23 Transfer 30 µl of supernatant from each well of the PAP plate to the corresponding well of
the new MIDI plate labeled with the FSP barcode.
SAFE STOPPING POINT
If you do not plan to proceed immediately to Validate Final Product on page 35, you can safely stop
the protocol here. If you are stopping, seal the FSP plate with a Microseal ‘B’ adhesive seal and
store at -25°C to -15°C for up to 7 days.
Comments
__________________________________________________________________________________
__________________________________________________________________________________
Page 34 of 36
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Validate Final Product
Perform the following procedures for quality control analysis on your sample library and
quantification of the final library.
Consumables
Item
Quantity
Storage
Supplied By
Resuspension Buffer (RSB)
1 tube
2°C to 8°C
Illumina
KAPA Library Quantification Kit Illumina/Universal
1
As specified by
manufacturer
User
High Sensitivity DNA Kit
1
As specified by
manufacturer
User
Qubit dsDNA HS Assay Kit
1
As specified by
manufacturer
User
Quantify Libraries
Illumina recommends that you quantify your libraries by qPCR.
Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina sequencing
platforms Technical Data Sheet using the KAPA standard, with the following modification:
Perform a size adjustment calculation to account for the difference in size between the average
fragment length of the library and the KAPA DNA Standard (452 bp). Determine the average
fragment length of the library between 200–2000 bp using an Agilent Technologies 2100
Bioanalyzer or equivalent. Use this average fragment length for the size adjustment calculation.
Quality Control
[_] 1
Dilute the Final DNA library from the FSP plate to an optimal concentration for the Agilent
Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip as follows:
[_] a Quant the Final DNA library using a Qubit dsDNA HS Assay Kit.
[_] b Dilute 2 µl of the Final DNA library to 1 ng/µl with Resuspension Buffer.
[_] 2
Load 1 µl of the diluted Final DNA library on an Agilent Technologies 2100 Bioanalyzer
using a High Sensitivity DNA chip.
[_] 3
Prepare a 1:5 dilution of the QC1:Pre-size selection DNA library, from step 20 of Pool and
Concentrate on page 31, with Resuspension Buffer.
[_] 4
Load 1 µl of the diluted QC1:Pre-size selection DNA library on an Agilent Technologies
2100 Bioanalyzer using a High Sensitivity DNA chip.
Check the size of the sample for a broad distribution of DNA fragments with a size range
from approximately 200–3000 bp.
[_] 5
Do one of the following:
• Proceed to cluster generation.
• Store the sealed FSP plate at -25°C to -15°C.
TruSeq Synthetic Long-Read DNA Library Prep
Experienced User Card
Part # 15047265 Rev. B
Page 35 of 36
Comments
__________________________________________________________________________________
__________________________________________________________________________________
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
*15047265*
Part # 15047265 Rev. B
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