Kirst’s Lab
Sequence Capture Protocol – Version 3
University of Florida
Method Overview:
This protocol describes the sample preparation for targeted sequencing on Pine megagametophytes. It is
based on Agilent’s SureSelect target enrichment method and sequencing with Illumina MiSeq, GAIIx or HiSeq2000.
I.)
Dissect haploid megagametophyte tissue from seed
Use new scalpel blades to open the seed coat. Tissue can be removed easier if seeds are stored overnight at -20oC
in autoclaved water. Be sure to avoid tissue contamination. Place individual megagametophytes in 2 mL centrifuge
tubes and freeze-dry them to completion (2+ hours).
II.)
Grind samples before starting DNA extraction
Place freeze-dried samples to Genogrinder with one stainless steel beads (OPS Diagnostics cat# GBSS 156-5000-01,
or similar). Grind for three minutes at medium 1X Rate (strokes/min).
III.)
DNA Extraction – Using DNeasy Plant Mini Kit, QIAGEN. Catalog # 69106 (with some changes):
1.) To each sample add Add 400 µL Buffer AP1 and 4 µL RNase A stock solution to each sample and VORTEX
vigorously (1 min). Do not mix Buffer AP1 and RNase A before use.
2.) Incubate the mixture for 10 min at 65 oC. Mix 2 or 3 times during incubation by inverting tube.
3.) Add 130 µL Buffer AP2 to the lysate, mix 30 sec (Vortex), and incubate for 5 min on ice.
4.) Centrifuge the lysate for 5 min at 14,000 rpm.
5.) Pipet the lysate (approx. 480 µL for loblolly pine megagametophyte) into the QIAsheredder Mini spin column
(lilac) placed in a 2 ml collection tube, and centrifuge for 2 min at 14,000 rpm.
6.) Transfer the flow-through fraction (approx. 460 µL) form step 5 into a new tube without disturbing the
cell-debris pellet.
7.) Add 1.5 volumes of Buffer AP3/E to the cleared lysate, and mix by pipetting. Note: it is important to pipet
Buffer AP3/E directly onto the cleared lysate and to mix immediately.
8.) Pipet 650 µL of the mixture from step 7, including any precipitate that may have formed, into the DNeasy Mini
spin column placed in a 2 ml collection tube. Centrifuge for 1 min at 14,000 rpm, and discard the flow-through.
Reuse the collection tube in step 9.
9.) Repeat step 8 with remaining sample. Discard the flow-through collection tube.
10.) Place the DNeasy Mini spin column into a new 2 ml collection tube, add 500 µL Buffer AW, and centrifuge for 1
min at 14,000 rpm. Discard the flow-through and reuse the collection tube in step 11.
11.) Add 500 µL Buffer AW to the DNeasy Mini spin column, and centrifuge for 2 min at 14,000 rpm to dry the
membrane.
12.) Transfer the DNeasy Mini spin column to a 1.5 ml or 2 ml microcentrifuge tube, and pipet 65 µL Low TE Buffer
onto the DNeasy membrane. Incubate for 5 min at room temperature (15-25 oC), and then centrifuge for 1
min at 14,000 rpm to elute.
13.) Repeat step 12 once, adding 60 µL Low TE Buffer instead of 65 µL. Note: 120 µL of the eluted sample are
going to be used on step V, the remaining 5 µL can be used for quality control (Nanodrop – Gel).
Low TE Buffer Recipe:
- 10 mM Tris_HCL pH 8.0
- 0.1 mM EDTA
* Samples can be stored at -20oC
Kirst’s Lab
IV.)
Sequence Capture Protocol – Version 3
University of Florida
Anneal adaptors (Aside step, can be placed in a different order)
First-time use: Prepare stock solution of the primers (500 µM)
-
Find the nmoles quantity of the primer.
Spin tubes. Add 2x as much TE as the nmoles value.
Vortex for 1 minute and spin. Store at -20oC. Pipette from stock using filtered tips.
Routine use: Prepare working solution of the primers (45 µM). Make sure barcodes are correct.
Example for 50 µL. Prepare it in 0.2 µL tubes.
-
Barcode X strand A at 500 µM
Barcode X strand B at 500 µM
10X NEB 2 buffer
H2O
2.25 µL
2.25 µL
5 µL
40.5 µL
Incubate in Thermocycler with the following program:
95oC
3 min
o
90 C
1 min
85oC
1 min
75oC
1 min
Decrease 1oC every 30 seconds until it reaches 4oC
V.)
Shearing Genomic DNA – Using Covaris E210 machine
A note about DNA input. DNA yield varies depending on megagametophyte size. We get an average of 1 µg
(Nanodrop based) but we have processed samples with input DNA ranging from 0.4 µg to 3 µg using this protocol.
If the concentration of the DNA sample is high, take 2 µg of DNA and dilute in Low TE Buffer to a final volume of
100 µl.
1.) Start the Covaris machine to 2 hours prior to preparing the samples to cool the water.
2.) Pipet 120 µL of each sample into a Covaris Micro Tube (6 x 16 mm), AFA Fiber with Snap-Cap round bottom
glass tube (cat # 520045). If working in a 96 well format, pipet 100 µl of the sample into a Covaris 96 Micro
Tube Plate (cat # 520078).
3.) Check treatment settings on the computer program:
a. Duty Cycle: 10%
b. Intensity: 5
c. Cycles/Burst: 200
d. Time: 180 seconds per sample
e. Water temp: Should be less than 8oC, usually 5oC.
4.) Load samples on tubes rack.
5.) On the computer program follow the next steps: Load Sample  Check aligment Start
6.) Shearing 96 samples takes 5 hrs.
Kirst’s Lab
VI.)
Sequence Capture Protocol – Version 3
University of Florida
DNA Purification – Using Agencourt AMPure XP Bead Purification Catalog # A63880.
A note about bead purification: The ratio of reaction to bead volume is usually 1.8x, unless otherwise stated.
Make sure to use a plate that seals very well to avoid cross-contamination while mixing the reactions. Use
standard centrifuge tubes for few samples.
For this step use 150 µL of beads for a 100 µl sample volume. Elute in 35 µl of EB buffer, recover 34 µl.
1.)
2.)
3.)
4.)
5.)
6.)
7.)
Let beads come to room temperature for 30 min. Resuspend beads well by vortexing.
Add 1.8x the reaction volume of beads. Seal plate well and mix the reaction.
Incubate for 5 min at room temperature.
Place plate in magnet until solution is clear (3-5 min).
Keep tubes in magnet. Collect solution with multichannel avoiding disturbing the pellet. Discard solution.
Prepare fresh 70% ethanol.
Keep tubes on magnet. Wash 2x with 200 µL ethanol 70%. Wait 30 sec for beads to settle after adding ethanol
and then remove it. Make sure to remove all remaining ethanol after the second wash.
8.) Wait 5 min for beads to dry (do not to over dry).
9.) Of the magnet, add 35 µL of EB buffer to elute (from Quiagen kit). Incubate for 2 min at room temperature.
10.) Place plate on magnet until solution is clear (2-3 min). Solution has DNA, move 34 µl to a new plate. 1 µl has to
be left behind to avoid carrying over beads.
EB Buffer Recipe:
- 10 mM Tris_HCL pH 8.5
* Samples can be stored at -20oC
VII.)
End Repair – Using End-ITTM, DNA End-Repair Kit EPICENTRE BIOTECHNOLOGIES Catalog # ER81050
1.) Prepare the following reaction mix - Master Mix (MM), values for one sample:
a. 10X End-Repair Buffer
5 µL
b. dNTP mix
5 µL
c. ATP
5 µL
d. End-Repair Enzyme Mix
1 µL
e. Total MM per sample:
16 µL
2.) Mix very well by pipetting. Add 16 µL of the mix to 30µL of each sample.
3.) Incubate at 25oC for 45 min (use Thermocycler).
4.) Purify DNA as Step VI using 90 µL of beads, eluting in 42 µL of EB buffer and recovering 41 µl for next step.
* Samples can be stored at -20oC
Kirst’s Lab
VIII.)
Sequence Capture Protocol – Version 3
University of Florida
Addition of Single <A> base
1.) Prepare the following MM, values for one sample:
a. Klenow buffer (10X NEBuffer 2)
5 µL
b. dATP (10mM) (Promega cat# U1205)
1 µL
c. KlenowFragment 3' to 5' exo- (5U/µL, NEB cat# M0212S)
3 µL
d. Total MM per sample:
9 µL
2.) Mix very well by pipetting. Add 9 µL of the mix to 41 µL of each sample.
3.) Incubate samples at 37oC for 30 min (use Thermocycler).
4.) Purify DNA as Step VI using 90 µL of beads, eluting in 30 µL of EB buffer and recovering 29 µl for next step.
* Proceed straight to adapter ligation. Stopping at this step is not recommended.
IX.)
Adaptor ligation - Using Fast-Link TM, DNA Ligation Kit EPICENTRE BIOTECHNOLOGIES Catalog #
LK6201H.
1.) Thaw adaptors on ice (~45 min)
2.) Prepare the following MM, values for one sample:
a. 10X Fast-Link Ligation Buffer
4 µL
b. ATP (10mM)
4 µL
c. Fast-Link DNA Ligase
1 µL
d. Total MM per sample
9 µL
3.)
4.)
5.)
5.)
6.)
Mix very well by pipetting. Add 9 µL of the mix to 29 µL of each sample.
Add to each DNA sample 2 µL of its respective adaptor (See step IV, oligo mix 45µM).
Incubate samples at 25oC for 15 min (use Thermocycler).
Purify DNA as Step VI using 72 µL of beads, eluting in 41 µL of EB buffer and recovering 40 µl.
Transfer 13 µL of the ligated DNA to a new plate for the following step. Store the rest (27 µl) at -20oC.
* Samples can be stored at -20oC
X.)
PCR Enrichment
A note about number of cycles and reaction input. The number of cycles needed depends on DNA input and
multiplexing level. For every different project we recommend a small factorial experiment testing number of cycles
versus reaction input. We recommend preparing 6 libraries up to this point and doing the following:
Factor A: Reaction input from the ligated DNA. Levels: 1, 1/2 and 1/3 of the volume.
Factor B: Number of cycles in the PCR. Levels: 6, 10 and 14 cycles (change if necessary).
The reaction below uses 1/3 of the volume and 10 cycles. That was optimum considering our input DNA and 8-plex
reactions. When testing very low number of cycles (<6), perform qPCR quantification (e.g. Kapa Biosynthesis
catalog # KK4824) to make sure enough PCR product incorporated the sequence added by the PCR primers that is
needed for the fragments to bind to the flow-cell.
Kirst’s Lab
Sequence Capture Protocol – Version 3
University of Florida
A note about primer purification. We have been doing reactions with custom primers ordered with Invitrogen
using standard desalt purification and it works fine. However, because the universal primers are used less, we have
ordered them PAGE purified, which guarantees that a larger percentage of the primers are full-length.
1.) Dilute Primers 1.01 PAGE and 2.01 PAGE to a working solution of 25 µM
2.) Prepare the following MM, values for one sample:
1.) 5X Cloned Phusion Buffer HF
10 µL
2.) PCR_Primer 1.01 PAGE
1 µL
3.) PCR_Primer 2.01 PAGE
1 µL
4.) 10 mM dNTP mix
1.5 µL
5.) Phusion polymerase (NEB #F-350)
1 µL
6.) H2O
22.5 µL
7.) Total MM per sample
37 µL
3.) Mix very well by pipetting. Add 37 µL of the mix to 13 µL of the sample.
4.) Run the following PCR cycle:
1.) 98oC

1 min
o
2.) 98 C

10 sec
3.) 65oC

30 sec
o
4.) 72 C

45 sec
5.) Go to 2

9 times (test number of cycles)
o
6.) 72 C

5 min
7.) 4oC

Hold
5.) Purify DNA as Step VI using 90 µL of beads, eluting in 31 µL of EB buffer and recovering 30 µl.
* Samples can be stored at -20oC
XI.)
Quantify using PicoGreen (Quant-it PicoGreendsDNA Assay Kit, Invitrogen Cat # P11496)
1.) Prepare 1 X TE Buffer (kit provides 20 X TE Buffer stock).
2.) Use the following 96 well plate: Costar 3915, Assay Plate, Flat Bottom, Non-treated, Non-sterile, Black
 Follow the next Plate Layout:
1
2
3
4
5
6
7
8
A
Std 1 Std 1
1
1
9
9
17
17
B
Std 2 Std 2
2
2
10
10
18
18
C
Std 3 Std 3
3
3
11
11
19
19
D
Std 4 Std 4
4
4
12
12
20
20
E
Std 5 Std 5
5
5
13
13
21
21
F
Std 6 Std 6
6
6
14
14
22
22
G
Std 7 Std 7
7
7
15
15
23
23
H
Std 8 Std 8
8
8
16
16
24
24
* Columns 1 & 2 for Standard DNAs and columns 3 to 12 for Samples.
** Two wells per sample to reduce pipetting variation.
9
25
26
27
28
29
30
31
32
10
25
26
27
28
29
30
31
32
11
33
34
35
36
37
38
39
40
12
33
34
35
36
37
38
39
40
Kirst’s Lab
Sequence Capture Protocol – Version 3
University of Florida
3.) Prepare the Standard DNA, use component C (λ DNA Standard):
a. Dilute to 2 ng/µL in a volume of 1.5 ml
b. Prepare the following 8 standards (change standard according to expected concentration if needed)
Rows Std To XX ng of DNA Take XX µL of 2 ng/µL Standard And add XX µL of TE Buffer
A
1
100
50
50
B
2
75
37.5
62.5
C
3
50
25
75
D
4
25
12.5
87.5
E
5
12.5
6.25
93.75
F
6
6.25
3.125
96.875
G
7
3.125
1.5625
98.4375
H
8
0
0
100
* Note that each standard will be repeated twice in the plate layout, columns 1 and 2.
4.) For columns 3 to 12:
a. Add 99 µL of 1X TE Buffer
b. Add 1 µL of the DNA sample according to the plate layout
At this point all wells must have 100µL of solution.
5.) Prepare diluted PicoGreen and add it to the plate.
a. Dilution rate 1:200 1 of Component A and 199 of 1X TE Buffer
b. Pipet 100 µL of the diluted PicoGreen solution to each well
6.) Take the plate to the reader (ICBR’s Spectromax M5 and software Soft Max Pro 5.3)
a. Open Soft Max Pro 5.3  Drawer opens
b. Put plate on top of violet adapter for Top Read
c. Press Drawer to close reader
d. Open project saved: Open  User files Matias *.ppr
e. Once a file is open, double click on “Plate 01” to Add/Remove Samples or to Edit Standards
f. Highlight samples (Columns 3 to 12)  Select “Unknowns-No Dilut”
g. Click on “Series”  Select “Top”  2 Replicates
h. Ctrl + Alt + Shift shows the standards
i. Click Ok  Hit Read
Expected Results:
a.
b.
c.
Check Standard curve: Regression model should be good  R2≈ 0.999
Open Standards: Coefficient of Variation (CV) should be low
Open Unknowns-No Dilut: CV should be low
Then, Save the results (*.pda) and Export file to a *.txt and save it to a flash drive.
Kirst’s Lab
XII.)
Sequence Capture Protocol – Version 3
University of Florida
Pooling DNA
1.) DNA pooling is done based on PicoGreen Mean concentration of each sample.
2.) Select 8 samples with different barcodes to create one pool. Note that you must have at least 10
µL of the sample
3.) Pool the samples according to sample whose PicoGreen concentration is minimum, for example,
the following table shows the resulting PicoGreen mean concentration for 8 samples:
Sample ID
146-6
123-5
112-4
108-2
113
178-1
14-9
125-4
Adaptor
(Barcode)
1
2
3
4
5
6
7
8
PicoGreen Mean
Concentration (ng/µL)
33.914
34.065
40.19
36.284
40.184
39.709
16.098
23.41
Note that sample 14-9 has the lowest
concentration.
Since we have 10 µL of each sample, the
DNA content centration for that sample is
≈ 160.98 ng. So, if we pool 150 ng of each
sample we will have a final Pool whose
concentration will be 1200 ng. The
minimum concentration of the pool
required for the hybridization is 500 ng. Having a little more is desired for a quality control on the
gel (step XIV).
Then, divide the ng of each sample by its mean PicoGreen Concentration, thus:
For sample 14-9 we have:
𝑉𝑜𝑙𝑢𝑚𝑒 (𝜇𝑙)𝑓𝑜𝑟 𝑝𝑜𝑜𝑙𝑖𝑛𝑔 =
XIII.)
150 𝑛𝑔
16.098 𝑛𝑔/𝜇𝑙
= 9.32 𝜇𝑙. Finally,
Sample ID
Adaptor (Barcode)
PicoGreen Mean Concentration (ng/µL)
µLfor pooling
146-6
123-5
112-4
108-2
113
178-1
14-9
125-4
1
2
3
4
5
6
7
8
33.914
34.065
40.19
36.284
40.184
39.709
 16.098
23.41
4.42
4.40
3.73
4.13
3.73
3.78
9.32
6.41
Quality control Gel:
For quality control, run a 1.5 % Agarose Gel on 0.5 X TE Buffer for 70 min at 86 volts. You are expected to get
similar results as the one presented next:
Kirst’s Lab
Sequence Capture Protocol – Version 3
University of Florida
You can also include next to the pool sample a couple of individual samples to see the distribution of their DNA
size.
XIV.)
Hybridization
Hybridization is made following “SureSelect Target Enrichment Protocol, Agilent Technologies”. Pages 33 to
52. Manual Part number G3360-90000.
Appendix 1 – list of validated barcode
Same length barcodes previously used in the lab.
GACT,CAGT,ACTG,AGTC,GTCA,CTGA,TCAG,TGAC
Different length barcodes currently being used in the lab and in this publication.
ACC,AGG,CAG,GAC,ACTG,AGTC,GCGT,GTAA,TGACG,CGCTT,CTAGC,TCAGA,GTCACA,TAACGA,TAGGAA,ATTGGAT
,CTGACGC,TCGAAGA