TITLE: Reverse transcription, primer pools preparation and multiplex PCR steps for DENV2 serotype
AUTHORS: Laís Ceschini, Carla Julia da Silva Pessoa Vieira, Gustavo Lima, Luisa Maria Inácio da Silva, Raul Emídio Lima, Tiago Graf, Gabriel Wallau

[DESCRIPTION]
This step-by-step protocol describes the cDNA synthesis, primer pools preparation and multiplex PCR conditions with the main goal to sequence the complete genome of DENV2 serotype strains.

[STEPS]
SECTION: Reverse transcription
1. Using a 2mL tube prepare the Mix 1 described below for 96 samples:

 
 
 
   Mix 1Reverse transcriptionVol. (1x)96 samples (+2 = 98 to keep some extra due to pipetting issues)Random Hexamers (50µM)1µL98µLdNTPs mix (10mM cada)1µL98µLTotal2µL194µL


 
 
 
SECTION: Reverse transcription
2. Using 0,2mL PCR tubes or 96 wells plates add 11-16µL of extracted RNA from RT-PCR positive samples. Add 2µL of Mix 1 to the tube/well and take it to the thermocycler with the following set up:
	
	65ºC ---- 5 minutes

SECTION: Reverse transcription
3. Take the tubes/wells to ice for 1 minute. (you can prepare a water bath with ice cubes to have a uniform temperature distribution)
SECTION: Reverse transcription
4. Using a 2mL tube prepare Mix 2:


 
 
   Mix 2Reverse TranscriptionVol.(1x)96 samples (+2 = 98 to keep some extra due to pipetting issues)5x SSIV Buffer4µL392µL100mM DTT1µL98µLRNaseOUT ouRNase Inhibitor 1µL98µLSSIV Reverse Transcriptase1µL98µLTotal7µL686µL

 
 
SECTION: Reverse transcription
5. Add 7µL of Mix 2 to the tubes containing the Mix 1 plus RNA and take it to the thermocycler following the set up below:

		Step1:
	42ºC ---- 50 minutes
	70ºC ---- 10 minutes
	  4ºC ---- Hold

SECTION: Reverse transcription
6. Store the cDNA at -20ºC.

Observation:. As a suggestion, to improve the final results only samples RT-PCR positive showing a Ct value of < 30 should be used for cDNA conversion and genomic amplification
SECTION: Pools of primers
7. Select two 0,6mL tubes for each pool. 

SECTION: Pools of primers
8. Using the original 100uM primer solution eluted individually, put them together following the table below containing each primer volume.

TABLE 1: Primers and pool order. 
 
     Primer SequenceTmConcentration inside of the pool *POOLDENV2_1_LEFTCAGTTGACACGCGGTTTCTCTC0,030uM10ul1DENV2_1_RIGHTTTAGGAAACGAAGGAACGCCAC0,030uM10ul1DENV2_3_LEFTTGGCGTTCCATTTAACCACACG0,030uM10ul1DENV2_3_RIGHTCGTTCCTATGGTGTATGCCAGG0,030uM10ul1DENV2_5_LEFTTGTGTGACGACGATGGCAAAAA0,015uM5ul1DENV2_5_RIGHTCCTTGCCATGYTTTCCTGTGTCA0,015uM5ul1DENV2_7_LEFTGCACAGGCAATGGTTCCTAGAC0,0075uM2,5ul1DENV2_7_RIGHTAGGGATCTTACATGGAGAACCGT0,0075uM2,5ul1DENV2_9_LEFTACAGAAAAAGATAGCCCAGTCAACA0,015uM5ul1DENV2_9_RIGHTGTCACGACTCCCACCAATACTAG0,015uM5ul1DENV2_11_LEFTCTGATGTGGAAACAAATAACACCAGA0,015uM5ul1DENV2_11_RIGHTCATGGACGGCTCTGTTGTCTTT0,015uM5ul1DENV2_13_LEFTACAGACCAGGCTACCATACACA0,015uM5ul1DENV2_13_RIGHTGCATGTTTCGTTCCTACTCGGG0,015uM5ul1DENV2_15_LEFTTGCAGCTGGACTACTCTTGAGA0,015uM5ul1DENV2_15_RIGHTAAAATGCTCACCATCCCGACTG0,015uM5ul1DENV2_17_LEFTAATCCTGTCAATAACAATATCAGAAGATGG0,030uM10ul1DENV2_17_RIGHTGCTTCCAGCCTCCTCCATATGA0,030uM10ul1DENV2_19_LEFTAGGAAAAGTTGTGGGTCTTTATGGT0,030uM10ul1DENV2_19_RIGHTACTGGTGATAGCAGCCTCATAGT0,030uM10ul1DENV2_21_LEFTTGGGTCACGGATTTTAAAGGGAA0,015uM5ul1DENV2_21_RIGHTGCTTCTTTCCAGTGTGCACAGT0,015uM5ul1DENV2_23_LEFTCCTTTGTGGACCTAATGAGAAGAGG0,015uM5ul1DENV2_23_RIGHTCGGCAGTTCACTGAGAGCATGA0,015uM5ul1DENV2_25_LEFTCCACACTGGATAGCAGCTTCAA0,030uM10ul1DENV2_25_RIGHTCCCAAGACCCATTAGCACTGT0,030uM10ul1DENV2_27_LEFTGGATGCTACTCACAAGTCAACCC0,015uM5ul1DENV2_27_RIGHTGAAAAGAGAAGTCCAGCTCCGG0,015uM5ul1DENV2_29_LEFTACTGAGATGGTTCGTCGAGAGA0,015uM5ul1DENV2_29_RIGHTGTGGATTCCTCACTAAGGCTCCT0,015uM5ul1DENV2_31_LEFTCGCAACATCGGAATTGAAAGTGA0,015uM5ul1DENV2_31_RIGHTCCAAGGCTGCATTGCTTCTCAC0,015uM5ul1DENV2_33_LEFTGCTTGGAGCACGCTTCTTAGAG0,015uM5ul1DENV2_33_RIGHTTGGGCTTCCATATTGGTGAAAGT0,015uM5ul1DENV2_35_LEFTAGAGGATGGAACGATTGGACACA0,015uM5ul1DENV2_35_RIGHTCCACTGGAGTTTTGTCTTCCATCC0,015uM5ul1DENV2_37_LEFTGAAGAGGAAGAGGCAGGWGTCC0,015uM5ul1DENV2_37_RIGHTCTGGAATGATGCTGAGGAGACAG0,015uM5ul1DENV2_2_LEFTATGCTGAAACGCGAGAGAAACC0,030uM10ul2DENV2_2_RIGHTCATGGCCATGAGGGTACACATG0,030uM10ul2DENV2_4_LEFTCATGGATGTCATCAGAAGGGGC0,015uM5ul2DENV2_4_RIGHTTCTGTTGTTGTGTTGGTCAGCT0,015uM5ul2DENV2_6_LEFTGGCATTGTGACCTGTGCTATGT0,015uM5ul2DENV2_6_RIGHTGGGGATTTTTGAAAGTGACCAATGT0,015uM5ul2DENV2_8_LEFTACAGCTCAAAGGAATGTCATACTCT0,015uM5ul2DENV2_8_RIGHTTCCTCCCAGGGATCCAAAATCC0,015uM5ul2DENV2_10_LEFTAGTGGGGTCTCATGGACTATGA0,015uM5ul2DENV2_10_RIGHTGATCGTTTTCCTGCCTGCATGA0,015uM5ul2DENV2_12_LEFTCCCAACACAAACAGAGCTTGGA0,0075uM2,5ul2DENV2_12_RIGHTTTTCCACAGTCCTCAGTCACCA0,0075uM2,5ul2DENV2_14_LEFTCGGACATGGGCAGATTGACAAC0,015uM5ul2DENV2_14_RIGHTCCAAGGCTAACGCATCAGTCAG0,015uM5ul2DENV2_16_LEFTGCAGAAAGCGGATTGGATACCA0,030uM10ul2DENV2_16_RIGHTATGCTGCTGCCGTGATTGGTAT0,030uM10ul2DENV2_18_LEFTAGATCGGAGCCGGAGTTTACAA0,030uM10ul2DENV2_18_RIGHTTGTCATCTTCGATCTCTGGATTGTC0,030uM10ul2DENV2_20_LEFTATACCAAACCCCAGCCATCAGA0,015uM5ul2DENV2_20_RIGHTCCTACTGAGTTGTATCACTTTCTTTCCA0,015uM5ul2DENV2_22_LEFTATGCCAGTGACCCACTCTAGTG0,015uM5ul2DENV2_22_RIGHTCCTTTCCCCTTCTTTTGTCCAGA0,015uM5ul2DENV2_24_LEFTTCCAACTTTCATGACTCAGAAGGC0,015uM5ul2DENV2_24_RIGHTGGAAACCCATCTCGTTTGCCAT0,015uM5ul2DENV2_26_LEFTACATCCTGGACATAGATCTACGTCC0,030uM10ul2DENV2_26_RIGHTAGGTCAATCACTGTTATTCCATCGAC0,030uM10ul2DENV2_28_LEFTTGTGGGAAGGAAATCCAGGGAG0,0075uM2,5ul2DENV2_28_RIGHTCCCCACAATAGTATGACCAGCC0,0075uM2,5ul2DENV2_30_LEFTAAGCAGGACGAACACTCAGAGT0,015uM5ul2DENV2_30_RIGHTCCCACGTTTTGTATGGGTGGTC0,015uM5ul2DENV2_32_LEFTGGCAGAGTGGCTTTGGAAAGAA0,015uM5ul2DENV2_32_RIGHTCCTTCTCCTTCCACTCCACTCA0,015uM5ul2DENV2_34_LEFTAAAGACCAACACCAAGAGGCAC0,015uM5ul2DENV2_34_RIGHTCAGTTCATCTTGGTTTCTGCATGG0,015uM5ul2DENV2_36_LEFTGAACAACCTGGTCCATACACGC0,0075uM2,5ul2DENV2_36_RIGHTGGGGCTCACAGGTAGCATAGTT0,0075uM2,5ul2

 
 Note: The primers were designed using the https://primalscheme.com based on the FJ467493, KY923048, KX274130, MG189962, KT187556, KU365903, KU517845, KY794785, KU948303, KU517847, KX372564, KX452038, KX380815, KU509277, KY627762, KY427085, KJ830750, MG779196, KY937188, EU660415, KF955402, MF459663, KU509273, FJ906969, KU094070, KM587709, HQ891023, HQ541799, HQ541798, KJ734727, KU509267, KJ189308, KY474331, KX702404, JX286526, KP188554, KP188555, JX669479, KP188551, KP188550  reference genomes.

SECTION: Pools of primers
9. Pool 1 will have a final volume of 230µl and pool 2 of 190µl.
SECTION: Pools of primers
10. In order to prepare the solution to use in the Multiplex PCR, dilute each pool 1:10. That is, 10µl of pool 1 and 90µl of ultrapure water.


SECTION: Multiplex PCR
11. Prepare the Mix 1 for a Multiplex PCR for each Pool 1 e Pool 2 using a Falcon tube of 15mL (~96 amostras) or a 2mL tube.

 
 
 
    Mix 1Multiplex PCRVol. Pool 1(1x)Vol. Pool 2(1x)96 amostras (+2) (pool1 ou pool2)Q5 Master Mix High fidelity 2X 12,5 µl 12,5 µl1.225 µlConjunto de primers (Pool1 ou Pool2) /concentração de uso/1,5 µl1,5 µl147 µlÁgua Ultra Pura8,5  µl8,5  µl833 µlTotal22,5µl22,5µl2205µl


 
 
 
SECTION: Multiplex PCR
12. Add  2,5µl of cDNA (totalling 5µl) in 22,5µl of the pool1 and pool2 reaction and take it to the thermocycler following the conditions bellow:
	
Step1:
98ºC ---- 30 seconds
Step2: (45 cycles)
98ºC ---- 15 seconds
58ºC ---- 30 seconds
72ºC ---- 5 minutes
Step3:
72ºC ---- 2 minutes
Hold 4ºC