Dec 19, 2023

Public workspaceTaME-seq2 V.2

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.dm6gpjxy5gzp/v2
  • 1Akershus University Hospital;
  • 2Oslo Metropolitan University;
  • 3University of Oslo
Alexander Hesselberg
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gpjxy5gzp/v2
Protocol Citation: jean-marc, Milan Stosic, Alexander Hesselberg, trinro 2023. TaME-seq2. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpjxy5gzp/v2Version created by Alexander Hesselberg
Manuscript citation:
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 Virol J 20, 44 (2023). https://doi.org/10.1186/s12985-023-02002-5
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 19, 2023
Last Modified: December 19, 2023
Protocol Integer ID: 92490
Keywords: integration, library preparation, illumina, virus, viral integrations, snp, snps, variants, whole genome sequencing, genome, genomics, enrichment, multiplex PCR
Abstract
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.
Materials

ABC
ComponentSupplierCatalog number
Illumina® DNA Prep, (M) Tagmentation (96 Samples)Illumina20018705
QIAGEN Multiplex PCR Kit (100)Quiagen206143
AMPure XP 60 mLBeckman Coulter A63881

Sample preparation
Sample preparation
Prepare and normalize DNA/cDNA samples by measuring sample concentration and diluting in nuclease-free water if necessary.

Sample volume should be Amount15 µL

Amount50 ng input is recommended, but the protocol works with less and performance is more dependent on viral load.

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


ABC
Reagent1x10x
Bead linked transposoms (BLT)5 μL50 μL
Tagmentation buffer 1 (TB1)5 μL50 μL
Sample DNA/cDNA15 μL
Total25 μL10x10 μL
Tagmentation reagent mix table

Note
You should prepare 10% extra to account for loss during pipetting

Incubate the samples as follows:
Duration00:15:00 at Temperature55 °C
Hold at Temperature4 °C
Note
Let it cool down to Temperature4 °C before taking it out of the thermocycler.


15m
Add Amount5 µL of Tagmentation stop buffer (TSB) to each sample.
Note
The Tagmentation stop buffer needs to be vortexed well before use.


Incubate sample for Duration00:15:00 at Temperature37 °C
Note
The samples can be stored in the fridge for a few hours after this step.


15m
Wash the samples

  1. Place tubes on the magnetic rack for Duration00:03:00 (or until solution is clear)
  2. Discard supernatant
  3. Remove tube from magnetic rack and add Amount50 µL Tagment wash buffer (TWB) and mix to resuspend beads
  4. Place tubes on magnetic rack for Duration00:03:00 and remove supernatant
  5. Repeat step 3 and 4 for a total of 2 washes
  6. Add Amount50 µL TWB to samples and mix
  7. Close cap on tubes and place on magnet. Allow to incubate for at least Duration00:03:00 and continue the protocol. The samples will be used later in the PCR
Note
The samples are submerged in TWB to stop the beads from overdrying while working on the next steps.

The samples can also be eluted in Amount14 µL nuclease-free water and stored in the freezer, if so do not add H2O/elution buffer to the PCR master mix in the next step.








9m
Amplification of tagmented DNA
Amplification of tagmented DNA

  1. Make the PCR master mix
ABC
Reagent1x10x
2x PCR master mix12,5 μl125 μl
Q-solution x52,5 μl25 μl
Primer pool (15 μM)1 μl
i5/i7 indexes (10 μM)2 μl
Nuclease-free water7 μl70 μl
Total25 μl22 μL x 10 μL + 3 μL primers per sample

Note
One master mix need to be prepared for each of the forward and reverse primer pools.

Nuclease-free water, primer pool and i5/i7 indexes will be dispensed in each individual well in the downstream PCR 96-well plate.

If samples were eluted in nuclease-free water after step 6, add Amount7 µL of the eluted sample to the individual wells instead of nuclease-free water.



2. Remove supernatant from samples prepared in step 6.7 and remove from magnetic rack
3. Add Amount30 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 Amount15 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 Temperature95 °C Duration00:05:00 1
Touchdown PCR:
Denaturation Temperature95 °C Duration00:00:30 10
Annealing Temperature68 °C -Temperature58 °C Duration00:01:30 10 (decrease Temperature1 °C per cycle)
Extension Temperature72 °C Duration00:00:30 10
PCR:
Denaturation Temperature95 °C Duration00:00:30 26
Annealing Temperature58 °C Duration00:01:30 26
Extension Temperature72 °C Duration00:00:30 26
Final extension Temperature68 °C Duration00:10:00 26
Hold Temperature4 °C


Note
We use a touchdown PCR as this gives the best amplification yield for us. This, and other PCR parameters, might differ between primer pools.

If amplification yield is low, consider changing the ramping temperature.

20m
Clean up and size selection, pooling and quality control
Clean up and size selection, pooling and quality control
5m
1) After the PCR reaction is finished place the plate on the magnet for Duration00:05:00
2) Transfer Amount20 µL of the supernatant to a fresh well (transfer less than the total volume to compensate for differences in volume during pipetting).
3) Pool Amount10 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).
AB
ReagentVolume per reaction
PB10 µL
Nuclease-free water8,85 µL

6) Vortex and mix the diluted master mix and add Amount18.75 µL (per reaction) master mix to the pool and mix well
Note
The mixing is essential for a good size selection and increasing yield

7) Seal the tubes and incubate at room temperature for Duration00:05:00
8) Place on magnet for Duration00:05:00 5 min or until clear
9) Add Amount3.7 µL (per reaction) of PB to a new eppendorf tube
10) Transfer Amount27 µL (per reaction) of the supernatant to the eppendorf tube containing PB and mix well
Note
The mixing is essential for a good size selection and increasing yield

11) Seal the tube and incubate at room temperature for Duration00:05:00
12) Place the tube on a magnet for Duration00:05:00 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 Duration00:01:00
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 Duration00:05:00 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 Amount205 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 Duration00:05:00
21) Place tube on magnet for Duration00:02:00 or until clear
22) Transfer Amount200 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 Amount42 µL and transfer Amount40 µL to a new tube before assessing size distribution again.
38m

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 Amount42 µL and transfer Amount40 µL to a new tube before assessing size distribution again.