TITLE: cDNA library preparation using ARTIC v3 primers and NEB UDI UMI adaptors for NGS
AUTHORS: Trevor Ho, Nahuel Manzanaro Moreno, Marion Walker, Nick Gilbert, Livia C T Scorza

[DESCRIPTION]
This is a cDNA library preparation protocol with the aim to quantify SARS-CoV-2 in wastewater and potentially detect its different variants using Next Generation Sequencing (Illumina). 

In this protocol, we amplified cDNA from viral RNA extracted from wastewater. 

The main steps involve:

Amplification of cDNA with ARTIC v3 primers
Ligation of amplified cDNA to NEB UDI UMI adaptors
PCR enrichment of the libraries
Pooling libraries together for sequencing

The further analysis pipeline used to perform assembly and intra-host/low-frequency variant calling is the nf-core/viralrecon:  https://nf-co.re/viralrecon

This protocol is still under revision

[STEPS]
SECTION: cDNA synthesis
1. 
 


For cDNA synthesis, refer to 

NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®) NEB #E7650S/L   

Chapter 2 Standard Protocol with cDNA Amplicon and Ligation Bead Cleanups (Three clean-up steps)

- Follow steps 2.1 to 2.3 

SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
4. For choosing adaptors, follow the following manual: 

NEBNext® Multiplex Oligos for Illumina® - Unique Dual Index UMI Adaptors DNA Set 1
NEB #E7395S 
  

"Designed for use in library prep for DNA, ChIP DNA and RNA (but not Small RNA), the NEBNext Unique Dual Index UMI (Unique Molecular Identifier) Adaptors enable high-efficiency adaptor ligation and high library yields. 
These adaptors contain all necessary sequences for sequencing on the Illumina platform, and are compatible with PCR-free applications and sample pooling prior to PCR amplification". 


SECTION: Cleanup of Adaptor-ligated DNA
9. As we know most amplicons are 400 bp in size, it is not necessary to use the size selection protocol.

Rather, we move to section 3B Cleanup of Adaptor-ligated DNA without size selection for all samples 

Protocol: NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® NEB #E7645S/L, #E7103S/L
 

 




SECTION: PCR enrichment
10. Follow on to Step 4. PCR Enrichment of Adaptor-ligated DNA
Protocol: NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® NEB #E7645S/L, #E7103S/L

Determine the number of cycles in your PCR reaction: 
 

SECTION: Checking library size distribution and library quantification
13. After clean up, run 0.5 µL of the cDNA (diluted 1:10  4.5 μL of water) on a Agilent TapeStation with HS D1000 Tapes to look at the size distribution. 


We recommend running a few randomly selected libraries to confirm size distirbution. 
If this the first time doing it and we see consistent aberrant patterns showing up, then it will be necessary to run all libraries to judge how widespread the issue is. 
 
Below is an example of expected size distribution

  

SECTION: 3.	Ligate amplified cDNA to NEB UDI UMI adaptors
9.1. 
SECTION: cDNA synthesis
2. After cDNA clean up, you can run 1 µL of the cDNA, diluted 1:10 on Agilent TapeStation with HS D1000 Tapes to look at the size distribution. 

If you are confident the sizes would be correct, just use Qubit HS dsDNA protocol to quantify the input DNA amount. 
 

SECTION: cDNA synthesis
3. Store the amplified cDNA at -20 °C.
SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
6. Check Index Pooling guidelines in the manual mentioned above (NEB #E7395S ) 

SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
5. Refer to tables 1-4 for information on index sequences 

SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
8. Adaptor dilution and ligation

In this step we use parts of both protocols  (E7395 and E7650) 
- Adaptors with UMI are used
- A smaller reaction volume is used following that of the ARTIC kit



SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
8.1. Adaptor dilution (optional depending on amount of input DNA)
In this step the cDNA quantification information is detrimental. Depending on the amount of DNA input, adaptor dilution is a necessary step. 

"If DNA input is ≤ 100 ng, dilute NEBNext Adaptor"

Please refer to NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®
NEB #E7645S/L, #E7103S/L   

Step 2. "Adaptor Ligation - Determine whether adaptor dilution is necessary"


SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
7. End Prep

Follow the step 2.4 of NEBnext End Prep  protocol in the NEB kit for SARS-CoV-2 detection manual (E7650)  
SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
8.2. Adaptor ligation

Use NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® NEB #E7645S/L, #E7103S/L as a guide


The E7650 protocol uses NEBNext adaptors for Illumina which are truncated and must be PCRed to add the full P5 and P7 sequences. 

Those NEBNext adaptors also need the USER enzyme because the adaptors need to be cleaved prior to the PCR step. 

The E7395 protocol does not require this.


Mixing together these features the ligation recipe was rewritten as follows: 
 
 
 
COMPONENTVOLUME (µL)10.4End Prep Reaction Mixture30-Diluted or undiluted NEBNext UMI Adaptors for Illumina1.25-(red) NEBNext Ultra II Ligation Master Mix15156(red) NEBNext Ligation Enhancer0.55.2Total Volume46.75


 
 
 
SECTION: PCR enrichment
11. Proceed to Step 4.1. PCR Amplification 

From NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® NEB #E7645S/L, #E7103S/L
SECTION: PCR enrichment
12. PCR enriched library clean up

Follow on to Step 5 of the same manual 

SECTION: Checking library size distribution and library quantification
14. Use a quantification method such as Qubit HS dsDNA protocol to quantify the input DNA amount. 
DNA quantification here is very important
SECTION: Pooling the libraries for sequencing
15. The samples that are going to be sequenced have to be pooled together in equimolar concentrations.
 

SECTION: Pooling the libraries for sequencing
16. 
 Example of how samples were prepared
 
Project library IDLibrary ID submitted to Edinburgh GenomicsSample IDcDNA sourceTotal library DNA amount (µg)Remaining volume (µL)Concentration (ng/µL)Volume ratio to reach lowest concentrationScaled volume to mix (µL)*Rounded volume (µL)DNA amount in mixed volume (ng)Mixed DNA concentration (ng/µL)Mixed DNA concentration (nM)CWW_NGS_l001NGS003_R1_p01_s01s001EPC1.994229.567.60.44526627226.23372781076.23421.148CWW_NGS_l002NGS003_R1_p01_s02s002SEPA Jan sample 2592.2272529.575.50.39867549675.58145695365.58421.29CWW_NGS_l003NGS003_R1_p01_s03s003SEPA Aug sample 6252.0384529.569.10.43560057896.09840810426.1421.51CWW_NGS_l004NGS003_R1_p01_s04s004SEPA Aug sample 6682.6579529.590.10.33407325194.67702552724.68421.668CWW_NGS_l005NGS003_R1_p01_s05s005Exeter sample WHIT 4/22.159429.573.20.41120218585.75683060115.76421.632CWW_NGS_l006NGS003_R1_p01_s06s006EtOH precipitated then 25-fold diluted Exeter samples3.36329.51140.26403508773.69649122813.7421.8CWW_NGS_l007NGS003_R1_p01_s07s007EtOH precipitated then 25-fold diluted SEPA (jan) samples3.510529.51190.25294117653.54117647063.54421.26CWW_NGS_l008NGS003_R1_p01_s08s008SEPA Jan pooled samples0.8879529.530.111414421.4CWW_NGS_l009NGS003_R1_p01_s09s009SEPA Aug sample 6511.185929.540.20.748756218910.482587064710.48421.296CWW_NGS_l010NGS003_R1_p01_s10s010SEPA Aug sample 4651.191829.540.40.74504950510.430693069310.43421.372Total volume70.54214.37659.7783829787164.6787409882

 
 
SECTION: Ligate amplified cDNA to NEB UDI UMI adaptors
8.3. Follow to steps 2.3 and 2.4 of the aforementioned manual (NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® NEB #E7645S/L, #E7103S/L) 
 
Samples can be stored at - 20 overnight