Alternative Whole Genome Sequencing

Reagents

1. 2xTn5 dialysis Bf(DF):               

2. 5X TAPS-MgCl2 Buffer

1500 ul 50 mM TAPS-NaOH at pH 8.5, 25 mM MgCl₂

3. Tn5 Transposase

Primers 

Tn5MErev, 5′-[phos]CTGTCTCTTATACACATCT-3′;

Tn5ME-A (Illumina FC-121-1030), 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′;

Tn5ME-B (Illumina FC-121-1031), 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′

Primer A = mix equal molar Tn5MErev with Tn5ME-A

Primer B = mix equal molar Tn5MErev with Tn5ME-B

Tn5 based tagmentation library prep protocol

Amounts are for 2 plates

Transposon Assembly *note that this step should not be scaled down too much-the lowest I’ve successfully tried uses 50uL Tn5*

1. Remove primers Tn5ME-A, Tn5ME-B, Tn5ME-rev, DF buffer, and Tn5 from freezer. Melt primers and buffer on heat block, mix by vortexing and spin. Leave Tn5 to melt on bench. Turn on 70° incubator.

2. Prepare primers A and B in 1.5mL tube:

27.5uL primer Tn5ME-A + 27.5uL primer Tn5ME-rev = primer A (55uL)

27.5uL primer Tn5ME-B + 27.5uL primer Tn5ME-rev = primer B (55uL)

3. Incubate at 70° for 1 min then place in ice.

4. In a new 1.5mL tube combine the following:

85uL Tn5 (64uM)

55uL primer A

55uL primer B

110uL 2x DF buffer

305uL

            Mix by pipetting.

5. Incubate at room temperature for 2 hours.

Tagmentation

1. Start thermocyclers so they are up to temp. Program: 55° 60m, 55° 60m for 15uL volume.

2. In new 2mL tube add:                                     (1x)

900uL H2O                                          4.5uL

400uL 5x TAPS buffer                         2uL

400uL 40% PEG                                   2uL

200uL Tn5 mix                                    1uL

DNA (10ng/uL)                                    1uL

1900uL mix

Mix by inverting. Aliquot 225uL into a strip of 8, and use strip to aliquot 9uL into 2 plates using multichannel. If DNA is of questionable quality the volume can be increased to 1.5 or more, just scale water accordingly.

3. Move to DNA bench. Spin thawed (can place in fridge overnight) DNA dilution plates. Add 1uL DNA to prepared plates of mix. Seal with film and spin.

4. Incubate in thermocycler 10min at 55°

5. Remove from thermocycler. Carefully remove film and add 2.5uL 0.2% SDS (prepared in a strip tube). Seal with new film and return to thermocyclers to incubate for 7 mins.

PCR Enrichment

1. Remove dNTPs and buffer from freezer and thaw, vortex, and spin. Index plates should be thawed in fridge for a few hours beforehand, then spin down. Enzyme should only be out of the freezer briefly.

2. In a 2mL tube mix the following:                    (1x)

576uL H2O                                          3uL

960uL 5x PCR buffer                           5uL

57.6uL dNTPs (10mM)                        0.3uL

38.4uL HiFi PCR enzyme                     0.2uL

1632uL

            Mix by inverting and spin.

3. Aliquot 200uL into an 8-strip. Move to DNA bench. Add 7.5uL of mix to plates from tagmentation (which have 12.5uL of product, so will end with 20uL).

4. Add index. Index plates are pre-prepared with equal amounts of both indices, so add 5uL of the mix (=2.5uL each index) at 10uM each. Be careful that plate positions match!

5. Cover PCR plates with lids (not film), spin, and place in thermocyclers for the following program:

72°       3min

98°       30sec

98°      30sec

63°       30sec

72°       3min

8°         hold

6. When complete freeze at -20° overnight or proceed to cleanup.

Bead Cleanup *be sure Tecan is booked if QC will be done*

1. Remove beads from cold room and let sit at RT for 30mins. Mix by inversion, light shaking before use. Pour about 2.6mL into boat.

2. Add 7.5uL (with filter tips) to each well. Cover with film and shake by hand until homogenous. Incubate at RT 10 mins. Spin briefly.

3. Place on magnet stands for 5 mins until all beads are on the sides.

4. Pipette 28uL to new plates. Pipette opposite the beads so as not to disturb them.

5. Add 4.5uL beads (stir beads with pipette tip first). Cover with film, shake, and incubate 10 mins at RT. Spin briefly.

6. Place on magnet stand for 5 mins.

7. While still on magnet, pipette out and throw away 22.5uL-don’t touch beads. About 10uL remains.

8. Add fresh 80% EtOH to boat and add 70uL to each. Place large stack of paper towels over the top and invert (towels, plate, and magnet) and shake. Leave upside down on towels for about a minute.

9. Repeat #8, but before dumping lift plate from magnet and allow all beads to drop to the bottoms of the tubes. Place back on magnets and leave for 3mins before dumping. Shake vigorously and leave plate and magnet on its side on the bench to dry for about 3 mins.

10.  Add 22uL sterile H₂O to each (from boat with multichannel)

11.  Cover with film, remove from magnets and shake by hand on trays vigorously. Spin briefly and put on yellow shaker 2 mins @ 2000. Incubate at RT 20mins. Spin.

12.  Place on magnets for 5 mins. Prepare new plates during incubation.

13.  Remove film and pipette 19uL into new plates. Seal with lids (can re-use from original PCR). Proceed to QC or place in cold room.

QC & Pool *before starting remove Qubit standards from cold room to equilibrate*

1. Mix 15mL Qubit buffer with 80uL HS reagent, mix in falcon tube and pour into boat. Pipette 70uL into each well of a black plate (for Tecan).

2. Add 1uL library and seal with film.

3. Shake on yellow shaker @ 1200 30-60sec. Spin briefly.

4. Take to plate reader in D9 corridor. Turn on machine and remove film from plate.

5. Open Magellan. Put plate in machine with A1 to upper left. Press button to close tray. Press green arrow twice to continue. Select “use predefined method” and users->yanjun->96-nostandard-final.mth, make selection, start.

6. Note 2 of the lowest and 2 of the highest readings. Write position and reading. Edit->copy to Excel, file->save as (to desktop). Next->finish->close tray and exit. Copy your worksheet to USB.

7. Bring plates back to lab. Label Qubit tubes for 2 standards and each of the 4 samples you took note of. Put 190uL of the buffer/reagent mix (From step 1) in the tubes for the standards and 129uL in the others.

8. Add 10uL of each standard to the tubes and all remaining (about 69uL) of the samples to appropriate tubes. Vortex and incubate 2 mins.

9. Run Qubit-read standards, samples, calculate stock with 1uL volume, write down concentrations.

10.  Create a standard curve using these readings for each plate to estimate concentration of all samples.

11.  Pool accordingly. Store pools in cold room or freeze at -20 if not doing wash within a day.

Post-pooling wash

1. Take beads out to bring to RT 30mins before starting. Mix thoroughly.

2. Calculate how much of each pool you need to start with 1000ng (adjust for how much you need to send for sequencing. You’ll end up with less than half of this starting concentration). Calculate how much beads you need for each pool (.45x and .3x)

3. Transfer calculated amount of each pool into 2mL tubes.

4. Add 0.45x AmpPure beads. Mix by hand until homogenous.

5. Incubate 10min RT. Put remaining pools back in cold room.

6. Place tubes on magnet for 5min. Label new 2mL tubes.

7. Transfer supernatant to new tubes. Pipette away from beads.

8. Add 0.3x beads. Mix until homogenous.

9. Incubate 10min at RT.

10.  Place on magnet 5min

11.  Pipette off supernatant. Leave tubes open on magnet.

12.  Wash beads with 500uL 80% EtOH. Leave a couple seconds and pipette off EtOH into waste.

13.  Let dry 5min at RT. Aliquot some sterile H₂O into a 2mL tube.

14.  Suspend beads in 100uL H₂O, vortex.

15.  Place on magnet 10min.

16.  Move supernatant to new tubes.

17.  Measure product on Qubit and Tapestation as desired. Adjust volumes to needed concentration via speedvac if needed. If too low can repeat this section with higher starting amount.

Appendix 1

1. 2xTn5 dialysis Bf(DF):                1L (H20 added to vol.)

100 mM Hepes, pH 7.2                      100 mL 1M or 23,83 g           

200 mM NaCl                                     11.69 g NaCl

0.2 mM EDTA                                    400 uL 500 mM

2 mM DTT                                          2 mL 1M

0.2% Triton X-100                             2 mL Triton X-100

20% Glycerol                                      252 g 100% Glycerol

2. 5X TAPS-MgCl2

50 mM TAPS-NaOH at pH 8.5, 25 mM MgCl₂

Appendix 2

Tn5MErev, 5′-[phos]CTGTCTCTTATACACATCT-3′;

Tn5ME-A (Illumina FC-121-1030), 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′;

Tn5ME-B (Illumina FC-121-1031), 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′

Primer A = mix equal molar Tn5MErev with Tn5ME-A

Primer B = mix equal molar Tn5MErev with Tn5ME-B

PCR index primer are following Nextera XT Index Kit v2 - Index 2 (i5/i7) Adapters, sequences can be found on page 14 of illumina adapter sequences.  You can either order the whole kit from Illumina or synthesis it your self.  I was using orders from IDT with standard desalting and it worked fine.