TITLE: SARS-CoV-2 McGill Nextera Flex sequencing protocol_Lunascript_ARTIC.V3_5uLRT
AUTHORS: Sarah J Reiling, Marie-Michelle  Simon, Anne-Marie  Roy, Shu-Huang  Chen, Ioannis  Ragoussis

[DESCRIPTION]
How the Nextera DNA Flex Assay Works 
The Nextera DNA Flex library prep kit uses a bead-based transposome complex to tagment genomic DNA, which is a process that fragments DNA and then tags the DNA with adapter sequences in one step. After it is saturated with input DNA, the bead-based transposome complex fragments a set number of DNA molecules. This fragmentation provides flexibility to use a wide DNA input range to generate normalized libraries of consistent tight fragment size distribution. Following tagmentation, a limited-cycle PCR adds Nextera DNA Flex-specific index adapter sequences to the ends of a DNA fragment. This step enables capability across all Illumina sequencing platforms. A subsequent Sample Purification Beads (SPB) cleanup step then purifies libraries for use on an Illumina sequencer. The double-stranded DNA library is denatured before hybridization of the biotin probe oligonucleotide pool. 

PCR Amplicons for Nextera Flex
When starting with PCR amplicons, the PCR amplicon must be > 150 bp. The standard clean up protocol depletes libraries < 500 bp. Therefore, Illumina recommends that amplicons < 500 bp undergo a 1.8 x sample purification bead volume ratio to supernatant during Clean Up Libraries on page 11. Shorter amplicons can otherwise be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end. 

More information can be found here: https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf



[STEPS]
SECTION: cDNA preparation
1. cDNA PREPARATION



Mix the following components in a 0.2 mL 8-strip tube or in a 96-well plate:

Component                                        Volume

LunaScript RT SuperMix (5x)          4 µL   
Nuclease-free water                         5 µL
Template RNA                                   11 µL
Total                                                    20 µL 
 

 

SECTION: cDNA preparation
2. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
SECTION: cDNA preparation
3. Incubate the reaction as follows:

 25 °C for 2 min
55 °C for 20 min 
95 °C for 1 min 
Place on ice for 1 min or store cDNA at -20C
SECTION: Primer pool preparation
4. PRIMER POOL PREPARATION

If required resuspend lyophilised primers at a concentration of 100 µM each


SECTION: Primer pool preparation
6. Generate primer pool stocks by adding 5 µL of each primer pair to a 1.5 mL Eppendorf labelled either “Pool 1 (100 µM)” or “Pool 2 (100 µM)”. Total volume should be 490 µL for Pool 1 (100 µM) and 490 µL for Pool 2 (100 µM). These are your 100 µM stocks of each primer pool.
 

SECTION: Primer pool preparation
7. Dilute this primer pool 1:10 in molecular grade water, to generate 10 µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination. 
 
 

SECTION: Multiplex PCR
8. MULTIPLEX PCR

In the extraction and sample addition cabinet add 5 µL RT product to each tube and mix well by pipetting.
 

SECTION: Multiplex PCR
9. In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:

Component                                                             Pool 1 [10 uM primer]        Pool 2 [10 uM]      
Q5 Hot Start High-Fidelity 2X Master Mix         12.5 µL                           12.5 µL            
Primer Pool 1 or 2                                                  3.7 µL                            3.7 µL             
Nuclease-free water                                             3.8 µL                             3.8 µL              
Total                                                                        20 µL                              20 µL                  

Add 5 ul RT product as mentioned in step 10. 
 

SECTION: Multiplex PCR
10. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
SECTION: Multiplex PCR
11. Set-up the following program on the thermal cycler:  

Step                        Temperature     Time                    Cycles

Heat Activation     98 °C        30 s    1
Denaturation         98 °C        15 s     36 
Annealing              63 °C        5 min     36
Hold                       4 °C            Indefinite              1
 

SECTION: PCR clean-up
12. PCR CLEANUP

Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single 1.5 mLEppendorf tube. 

SECTION: PCR clean-up
13. Clean-up the amplicons using the following protocol:

Add a volume ratio of 0.8x of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
Incubate for 5 min at room temperature. 
Pellet on magnet for 5 min. Remove supernatant.
Add 200 ul of 80% ethanol to the pellet and wash twice. 
Elute in 30 ul elution buffer.

 

SECTION: Amplicon Quantification and normalisation
14. AMPLICON QUANTIFICATION AND NORMALIZATION

Quantify the amplicon pools using a fluorimetric dsDNA assay.  

We expect following concentrations:

Pool 1+2 combined:
100-150 ng/ul for Ct 14-24
30-80 ng/ul for Ct 25-29
10-30 ng/ul for Ct 30-36
SECTION: Amplicon Quantification and normalisation
15. Use the 30 ul with 150 ng DNA for Nextera Flex Tagmentation. 
Protocol can be found here:
https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nextera_dna_flex/nextera-dna-flex-library-prep-reference-guide-1000000025416-07.pdf

Start on page 7 with the Tagmentation.

SECTION: Tagment Genomic DNA
16.   
  
 





SECTION: Post Tagmentation Cleanup
17.   
  

SECTION: Amplify Tagmented DNA
18.   
  
  
 


19.   
  
  
 


SECTION: Pool Libraries
20.   

SECTION: Check Library Quality
21.   

SECTION: Dilute Libraries to the Starting Concentration
22.   
  

SECTION: Primer pool preparation
5. ARTIC nCov-2019  only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. Primer names and dilutions are listed in the table below. https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed