Possible Commercial Tn5 WGS Alternative

Original Commercial Protocol

Embody Protocol for Tagmentation Step 3 to the end of Bead Cleanup

Investment Costs

Preparation of Adapter Mix

1. The name and sequence of reference primers for Illumina platform: Primer A: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’
   Primer B: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’ Primer ME: 5'-pCTGTCTCTTATACACATCT-3

2. Dissolve Primer A, Primer B, Primer ME with Annealing Buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH 7.5) to 100 μM

3. Prepare the following reaction systems:

4. Mix the reaction 1 and reaction 2 thoroughly by vortexing, and briefly centrifuge to collect the solution to the bottom of the tube. Place the tubes in Thermocycler and perform the following program:

5. After the reaction, mix Reaction 1 and Reaction 2 in an equal volume, named Adapter Mix. Store at -20°C.

Preparation of Transposon

1. Prepare the following components to a sterile PCR tube:

2. Mix thoroughly by pipetting 20 times.

3. Incubate at RT (25°C) for 1 h, the obtained transposon can be directly used for DNA

   tagmentation, or stored at -20°C.

DNA Tagmentation

1. Thaw each component at room temperature, mix upside down before use.

2. Prepare the following components to a sterile PCR tube:

From here down it is the Embody until end of Bead Cleanup

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/Stop Solution (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:        

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:

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

Bead Cleanup

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 (Using plate reader and qPCR)

1. Use Take 3 Trio to quantify all products

2.  Identify the highest, lowest, and middle concentrations by absorbance

3. Quantify these by qPCR

4. check these for leftover primer dimers or other undesirables by tape station

5. Use these as standard curve to calculate molarity of all

6. Use normalization protocol to normalize

7. Pool appropriately