TITLE: TaME-seq2
AUTHORS: jean-marc, Milan Stosic, Alexander Hesselberg, trinro

[DESCRIPTION]
Tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling (TaME-seq2). Viral genome and integration enrichment library preparation protocol. 

Manuscript:
TaME-seq2: Tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling. 
Alexander Hesselberg Løvestad, Milan S. Stosic, Jean-Marc Costanzi, Irene Kraus Christiansen, Hege Vangstein Aamot, Ole Herman Ambur, Trine B. Rounge medRxiv 2022.12.22.22283851
doi: https://doi.org/10.1186/s12985-023-02002-5

Before starting: 
Prepare samples consisting of extracted DNA or reverse transcribed cDNA. 

The method consists of three main parts: 

Part 1: 
Tagmentation of samples using the Illumina DNA Prep kit. 

Part 2: 
Multiplex PCR reaction of tagmented DNA/cDNA, using virus-specific primers and i5/i7 indexes. 

Part 3:
Clean-up and size selection using purification beads/Ampure XP beads. 

[STEPS]
SECTION: Sample preparation
1. Prepare and normalize DNA/cDNA samples by measuring sample concentration and diluting in nuclease-free water if necessary. 

Sample volume should be 15 µL 

50 ng  input is recommended, but the protocol works with less and performance is more dependent on viral load. 

SECTION: Tagmentation
2. Prepare a master mix for the tagmentation reaction and add sample DNA and master mix to individual wells. 


 

 

SECTION: Tagmentation
3. Incubate the samples as follows:
 15 min at 55 °C 
Hold at 4 °C 
 

SECTION: Tagmentation
4. Add 5 µL  of Tagmentation stop buffer (TSB) to each sample.
 


SECTION: Tagmentation
5. Incubate sample for 15 min  at 37 °C 
 


SECTION: Tagmentation
6. Wash the samples

Place tubes on the magnetic rack for 3 min  (or until solution is clear)
Discard supernatant
Remove tube from magnetic rack and add 50 µL Tagment wash buffer (TWB) and mix to resuspend beads 
Place tubes on magnetic rack for 3 min and remove supernatant
Repeat step 3 and 4 for a total of 2 washes
Add 50 µL TWB to samples and mix
Close cap on tubes and place on magnet. Allow to incubate for at least 3 min  and continue the protocol. The samples will be used later in the PCR 
 







SECTION: Amplification of tagmented DNA
7. 
Make the PCR master mix 
 

 


2. Remove supernatant from samples prepared in step 6.7 and remove from magnetic rack
3. Add 30 µL of PCR MM to the samples, mix well and resuspend the beads (the beads can be difficult to resuspend, but they don't need to be completely respuspended)
4. Pipette 15 µL out of the sample to a new well containing the nuclease-free water, primer pool and i5/i7 indexes, for seperate F and R reactions
5. Run the touchdown PCR reaction using the following program on the thermal cycler: 

         Step                    Temperature                         Time            Cycles

Initial denaturation        95 °C                     5 min       1
Touchdown PCR:
Denaturation                 95 °C                     30 s       10 
Annealing              68 °C-58 °C          90 s        10 (decrease 1 °C per cycle)
Extension                      72 °C                      30 s       10
PCR:
Denaturation                95 °C                     30 s       26 
Annealing                      58 °C                     90 s        26
Extension                      72 °C                     30 s       26
Final extension             68 °C                     10 min        26
Hold                              4 °C                       


 

SECTION: Clean up and size selection, pooling and quality control
9. 
Assess the quality of the pools using a Bioanalyzer or an equivalent instrument to see the fragment size distribution. 

If there is an excess of small fragments in the library, clean up one or more times (start from step 11) using 0,65x ratio Ampure beads, elute in 42 µL and transfer 40 µL to a new tube before assessing size distribution again. 
SECTION: Clean up and size selection, pooling and quality control
8. 1) After the PCR reaction is finished place the plate on the magnet for 5 min
2) Transfer 20 µL of the supernatant to a fresh well (transfer less than the total volume to compensate for differences in volume during pipetting).
3) Pool 10 µL of each sample in an eppendorf tubes.
4) Vortex and invert purification beads (PB) to fully resuspend.
5) Prepare at master mix of diluted PB (Note: The volumes used in the master mix is for each sample that has been pooled). 
 

6) Vortex and mix the diluted master mix and add 18.75 µL (per reaction) master mix to the pool and mix well
 

7) Seal the tubes and incubate at room temperature for 5 min
8) Place on magnet for 5 min  5 min or until clear 
9) Add 3.7 µL (per reaction) of PB to a new eppendorf tube
10) Transfer 27 µL (per reaction) of the supernatant to the eppendorf tube containing PB and mix well
 

11) Seal the tube and incubate at room temperature for 5 min
12) Place the tube on a magnet for 5 min or until clear
13) Remove and discard the supernatant without disrupting the beads
14) With the tube on the magnet, add fresh 80% ethanol to cover the beads without mixing and incubate 1 min 
15) Remove the ethanol
16) Repeat steps 14 and 15 for a total of 2 washes
17) Remove any excess liquid from the tube
18) Air-dry on the magnet for 5 min or until dry. The bead should not dry so long that it cracks while ethanol residues should have evaporated. 
19) Remove the tube from the magnet and add 205 µL (more or less depending on desired reaction volume to elute in) resuspension buffer/water-free nuclease to the beads and mix
20) Incubate at room temperature for 5 min 
21) Place tube on magnet for 2 min or until clear
22) Transfer 200 µL of the supernatant into a fresh tube
23) Clean up two more times (start from step 12) using 0,65x ratio Ampure beads, elute in 42 µL and transfer 40 µL to a new tube before assessing size distribution again.