TITLE: A SARS-CoV-2 Synthetic Control Mixture Preparation Protocol 
AUTHORS: Jannatul Ferdous, April N Harris, wtaylo, Jessica A Schlueter, Cynthia Gibas

[DESCRIPTION]
Wastewater-based sequencing surveillance has been a powerful tool for monitoring SARS-CoV-2 and making public health decisions. Along with sequencing wastewater as a medium, this approach has additional complexity in the deconvolution process. The evaluation of existing bioinformatic deconvolution tools with a dataset with known composition and concentration is expected to aid in litigating the complexity of deconvolution. This is a protocol for generating a golden standard dataset with synthetic RNA control mixtures that is reproducible , easy to adapt to any other synthetic controls and sequencing platform and can be applied to performance evaluation studies.  


[GUIDELINES]
Always keep the RNA controls in -80 freezers if not using immediately. 
Plan ahead to work with these controls to minimize the freeze and thaw cycle. 

[STEPS]
SECTION: Selection of Synthetic Controls
2. Synthetic controls chosen for Ferdous et al. 2023  - Control 15 (Alpha-103909), Control 17 (Gamma-104044), Control 23 (Delta-104533), Control 48 (Omicron - BA.1 lineage-105204), Control 51 (B.1.1.529+BA.2-England-105346), Control 2 (Wuhan hu-1 from china-102024), Control 6 (Wuhan hu-1 from California-102918), Control 50 (B.1.1.529+BA.2-Australia-105345), Control 64 (BA.5-England-106196), Control 62 (B.2.12.1-Denmark-105865), Control 66 (BA.4-Texas-106198), Control 67 (BA.4-California-106199), Control 63 (B.2.12.1-USA-105857), Control 65 (BA.5-USA-106197) and Control 19 (Iota-104529). 
SECTION: Dilution of Synthetic Controls
4. Dilution can be modified based on the specific concentration requirement of the research design. For this dataset, dilute the controls in a 1:100 ratio as each control is provided in a standard format, which consists of 100ul at a concentration of approximately one million copies per microliter. 
SECTION: Creating Proper Documentation
10. It is crucial to maintain proper documentation while making the control mixture to make sure that the ground truth is completely error-free.
SECTION: Preparation of Synthetic Control Mixtures
14. Take out all the diluted controls from the freezer and thaw them in room temperature. 
SECTION: Selection of Synthetic Controls
1. Synthetic RNA controls were selected from Twist Biosciences (TwistBioscienceSyntheticViralControl). For this project, 15 different synthetic controls were chosen, however, any combination of controls can be ordered and combined for a similar analysis.
SECTION: Dilution of Synthetic Controls
5. Take 15 1.5ml eppendorf tubes and sterilize them with UV radiation.
SECTION: Dilution of Synthetic Controls
6. Label  the tubes as "Diluted Control x-date" with respective control name and date.  
SECTION: Dilution of Synthetic Controls
7. Add 99ul Nuclease free water to the tubes.
Add 1ul of each control in respective tubes . 
SECTION: Dilution of Synthetic Controls
8. Mix properly with gentle vortexing. 
SECTION: Dilution of Synthetic Controls
9. This diluted controls will be used as our stock to make control mixtures. Store the controls in -80 unless used immediately. 
SECTION: Selection of Synthetic Controls
3. Store the controls in -80 unless used immediately. It is crucial to minimize the number of freeze and thaw cycles with these RNA controls. So plan accordingly.
SECTION: Creating Proper Documentation
12. Fill in the information accordingly. 
SECTION: Creating Proper Documentation
11. We created a spreadsheet with columns "Mixture name"(Name of the mixture), "Controls"(controls that are added in this mixture), and "Amount"(Amount of each control added in this mixture). 
SECTION: Preparation of Synthetic Control Mixtures
15. Take 45 eppendorf tubes and decontaminate them with UV. 
Label them as the Mixtures names and organize them in eppendorf tube holder. 
SECTION: Preparation of Synthetic Control Mixtures
16. As per the spreadsheet above, take the tube of Mixture "All" and add all the control in 0.5ul amount.
Add 0.5 ul of NFW to that solution as well. 
Remember to change tips in between adding controls. 
SECTION: Preparation of Synthetic Control Mixtures
17. Close the lid and mix by gentle vortexing. 
Mixtue "All" is now completely prepared. 
SECTION: Preparation of Synthetic Control Mixtures
18. Prepare rest of the mixtures as per the spreadsheet. 

SECTION: Preparation of Synthetic Control Mixtures
19. Store all the control mixtures in -80 unless used immediately. 
SECTION: Sequencing
20. Follow the standard procedure of Oxford Nanopore sequencing library prep and loading (Protocol ) for sequencing the synthetic control mixtures.

SECTION: Sequencing
21. This can be modified according to the preferred choice of primers and sequencing platform.
SECTION: Creating Proper Documentation
13. Use this spreadsheet as a guideline while preparing the controls in the lab-ControlMixtureSpreadsheet