Reverse transcription, primer pools preparation and multiplex PCR steps for DENV1 serotype for genomic sequencing

Laís Ceschini; Carla Julia da Silva Pessoa Vieira; Luisa Maria Inácio da Silva; Gustavo Lima; Raul Emídio; Tiago Graf; Gabriel Luz Wallau

Aug 11, 2023

Reverse transcription, primer pools preparation and multiplex PCR steps for DENV1 serotype for genomic sequencing

DOI

dx.doi.org/10.17504/protocols.io.5jyl8pjyrg2w/v1

Laís Ceschini¹,
Carla Julia da Silva Pessoa Vieira¹,
Luisa Maria Inácio da Silva¹,
Gustavo Lima²,
Raul Emídio²,
Tiago Graf³,
Gabriel Luz Wallau¹

¹Departamento de Entomologia, Instituto Aggeu Magalhães (IAM);
²Núcleo de Plataformas Tecnológicas (NPT), Instituto Aggeu Magalhães (IAM);
³Instituto Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador

Luisa Maria Inácio da Silva

DOI: dx.doi.org/10.17504/protocols.io.5jyl8pjyrg2w/v1

Protocol Citation: Laís Ceschini, Carla Julia da Silva Pessoa Vieira, Luisa Maria Inácio da Silva, Gustavo Lima, Raul Emídio, Tiago Graf, Gabriel Luz Wallau 2023. Reverse transcription, primer pools preparation and multiplex PCR steps for DENV1 serotype for genomic sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pjyrg2w/v1

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: August 10, 2023

Last Modified: August 11, 2023

Protocol Integer ID: 86328

Funders Acknowledgements:

FIOCRUZ

Abstract

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 DENV1 serotype strains.

Materials

Reagents:

Reverse transcription: SuperScript™ IV First-Strand Synthesis System. (200 reactions) Cat:
18091200 Invitrogen

Multiplex PCR: Q5® High-Fidelity 2X Master Mix. Cat: M0492L NEB, H20 Ultre Pure,
primers described in table 1.

Reverse transcription

 Using a 2mL tube prepare the Mix 1 described below for 96 samples:



ABC
Mix 1
Reverse transcriptionVol. (1x) 96 samples (+2 = 98 to keep some extra due to pipetting issues)
Random Hexamers (50µM) 1µL 98µL
dNTPs mix (10mM each) 1µL 98µL
Total2µL194µL

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

Take the tubes/wells to ice for 1 minute (you can prepare a water bath with ice cubes to have a uniform temperature distribution).

Using a 2mL tube prepare Mix 2:


ABC
Mix 2
Reverse TranscriptionVol.
(1x)96 samples (+2 = 98 to keep
some extra due to pipetting
issues)
5x SSIV Buffer 4µL392µL
100mM DTT1µL98µL
RNaseOUT or
RNase Inhibitor1µL98µL
SSIV Reverse Transcriptase1µL98µL
Total7µL686µL

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

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.

Pools of primers

Select two 0,6mL tubes for each pool.

Using the original 100uM primer solution eluted individually, put them together following the table below containing each primer volume.

Pool 1 will have a final volume of 130µl and pool 2 of 170µl.

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.

TABLE 1: Primers and pool order


ABCDEF
PrimerSequenceTmConcentratio
n inside of
the pool *Volume of
primer within
the poolPOOL
DENV1SA_1_LEFTTACGTGGACCGACAAGAACAGT58.1ºC0,0075uM 2,5µl 1
DENV1SA_1_RIGHTACTATCATRTGTGGCTCTCCCC57.2ºC0,0075uM2,5µl1
DENV1SA_3_LEFT CACACGTGGGACTTGGTCTAGA58.4ºC  0,01125uM7,5µl 1
DENV1SA_3_RIGHTACACACAAAGTTCGCGTCTTGT57.6ºC 0,01125uM7,5µl 1
DENV1SA_5_LEFTCCTCACATTGGACTGCTCACCT59.1ºC0,01125uM7,5µl 1
DENV1SA_5_RIGHTTGCACTARRACAGTTCCATGCT56.6ºC0,01125uM7,5µl 1
DENV1SA_7_LEFT CGAGGAGCACGAAGGATGGC60.6ºC0,0075uM 2,5µl1
DENV1SA_7_RIGHTATGATGTTCTCAAGACGCGTGG57.5ºC 0,0075uM 2,5µl1
DENV1SA_9_LEFTTGGGAAGTTGAGGACTAYGGGT58.7ºC0,015uM5µl1
DENV1SA_9_RIGHTTGTRGTTCTGAGRGATGGACCTC57.8ºC0,015uM5µl1
DENV1SA_11_LEFT GATGACTGGAACACTGGCTGTT57.4ºC 0,030uM 10µl1
DENV1SA_11_RIGHDENV1SA_11_RIGHTCACCGGAAGCCATGTTGTTTTT56.7ºC0,030uM 10µl1
DENV1SA_13_LEFTAASAAGAAGCAGAACACTCCGG57.3ºC0,015uM 5µl1
DENV1SA_13_RIGHTACTGGCCCAGCTTGGTTCCAG62.4ºC0,015uM 5µl1
DENV1SA_15_LEFTATGGAGTGGTGACAACAAGTGG57.4ºC0,015uM 5µl1
DENV1SA_15_RIGHTGCTGGATCGGTAAARTGTGCTTC57.4ºC 0,015uM 5µl1
DENV1SA_17_LEFTACGGGTRATYCAAYTGAGCAGRA58.5ºC0,01125uM7,5µl1
DENV1SA_17_RIGHTCCTCTTCTCATGAGCTCCACA56.3ºC 0,01125uM7,5µl1
DENV1SA_19_LEFTAGTGTCTCAGGTGACCTAATATTGGA57ºC 0,0075uM2,5µl1
DENV1SA_19_RIGHTRGCTGCCACTGTCAGTATCATG57.5ºC 0,0075uM2,5µl1
DENV1SA_21_LEFTYGCAAAYCAGGCWGCYATATTGAT57.6ºC0,015uM5µl1
DENV1SA_21_RIGHTGATGTTTGCCATGGACACTGCT58.2ºC0,015uM5µl1
DENV1SA_23_LEFTACAACCAAACATGCAGTGTCGA57.5ºC0,015uM 5µl1
DENV1SA_23_RIGHTTTTCGCACTAGCATCCCTCCAT58.5ºC0,015uM 5µl1
DENV1SA_25_LEFT ACCTAGATATYATTGGCCAGAGGA55.9ºC 0,030uM10µl1
DENV1SA_25_RIGHTACCTTTCGTCTTCCACTGCTTC57.3ºC0,030uM10µl1
DENV1SA_27_LEFTTGGAAGGAGAAGGACTGCACAA58.4ºC 0,030uM 10µl1
DENV1SA_27_RIGHTCACRCAATCATCTCCGCTRATT55.5ºC 0,030uM 10µl1
DENV1SA_29_LEFTATGGAGCCTGAGAGAAACTGCT58.2ºC0,030uM 10µl1
DENV1SA_29_RIGHTGCYCCTTCGGGATCACTCTCAT59.7ºC0,030uM 10µl1
DENV1SA_2_LEFTTGTTGAACATAATRAACAGGAGGAA
AAGA 55.9ºC0,01125uM7,5µl2
DENV1SA_2_RIGHTGAATCCTGGGTGTCKCAAAGCC59.5ºC0,01125uM7,5µl2
DENV1SA_4_LEFTACTGTGCATTGAAGCTAAAATATCAA
ACA56ºC0,015uM 5µl2
DENV1SA_4_RIGHTACCATTGTTTGTGGACAAGCCA57.7ºC0,015uM5µl2
DENV1SA_6_LEFTAAACTGACYTTARAGGGGATGTCAT56.1ºC0,015uM5µl2
DENV1SA_6_RIGHTATATGCRGTCCCAAAAACCTGG56.7ºC0,015uM 5µl2
DENV1SA_8_LEFTAGGCTGACTCCCCAAAAAGACT58.5ºC0,015uM5µl2
DENV1SA_8_RIGHTTTGATGGCAGCTGACATTAGCC57.8ºC0,015uM5µl2
DENV1SA_10_LEFTGCAGGGCCATGGCACCTAGG63.5ºC0,0075uM 2,5µl 2
DENV1SA_10_RIGHTTCCCCATCCTGTCTGAAGCATT58.4ºC0,0075uM 2,5µl 2
DENV1SA_12_LEFTGGATTATGCATGGAARACAAYGGC56.9ºC0,030uM10µl2
DENV1SA_12_RIGHTGTGAGTGTRTCATCCCTYTCTTCA56.2ºC0,030uM10µl2
DENV1SA_14_LEFTAGGTCCCAAGTAGGAGTGGGAGT61.2ºC0,015uM 5µl2
DENV1SA_14_RIGHTCACCTCRTCCTCAATCTCTGGT57.2ºC0,015uM 5µl2
DENV1SA_16_LEFTGGGAGATAGTTGACCTCATGTGCCA60.3ºC0,015uM5µl2
DENV1SA_16_RIGHTCCTGTCGGCCCGGAAATTTGC61.7ºC0,015uM5µl2
DENV1SA_18_LEFTCAGAAGGGATCATCCCAGCCCT60.9ºC0,0075uM2,5µl2
DENV1SA_18_RIGHTCCTCCTTGTTCGGAATTGTGCA57.9ºC0,0075uM2,5µl2
DENV1SA_20_LEFTGCTGCTCATTCCAGARCCAGAC59.2ºC0,015uM5µl2
DENV1SA_20_RIGHTATGGGTTCACCTGGGAATAGCA58.4ºC0,015uM5µl2
DENV1SA_22_LEFTTCCATCACACTGGCTACTGGAC58.6ºC0,015uM 5µl2
DENV1SA_22_RIGHTCCCACAACCGAGGTCTATGACT58.4ºC0,015uM 5µl2
DENV1SA_24_LEFTGCTYAGAGGAAACCAATTCTGCA56.5ºC0,030uM10µl2
DENV1SA_24_RIGHTTGATCCTGATGGYTTGACCTCA54.7ºC0,030uM10µl2
DENV1SA_26_LEFTCTGCACAAGAGAGGAGTTCACA56.8ºC0,015uM5µl2
DENV1SA_26_RIGHTTATTCTTGTGTCCCATCCGGCT58.3ºC0,015uM 5µl2
DENV1SA_28_LEFTGAAACCCCCAAYCTAGCTRAGA56.4ºC0,030uM 10µl2
DENV1SA_28_RIGHTTAGCCGCTAGTCTCAGGTCTCT58.8ºC0,030uM 10µl2
DENV1SA_30_LEFTGGGCCACYAATATACAAGTAGCCA57.6ºC0,030uM 10µl2
DENV1SA_30_RIGHTCCCGCTGCTGCGTTATGTCT60.4ºC0,015uM 10µl2
DENV1SA_31_RIGHTCCTGTTGATTCAACAGCACCATTCCA59.7ºC0,015uM 10µl2
*approximate concentration of each primer in the 25µl PCR reaction.

Note: The primers were designed using the https://primalscheme.com (Brito, 2021) based on
the JX669463.1 and KP188568 reference genomes.

Multiplex PCR

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


ABCD
Mix 1
Multiplex PCR
Vol. Pool 1
(1x)Vol. Pool 2
(1x)96 samples (+2) (pool1 or pool2)
Q5 Master Mix High
fidelity 2X12,5 µl 12,5 µl1.225 µl
Pool primers (Pool1 ou
Pool2) /Use concentration/1,5 µl 1,5 µl147 µl
Ultra Pure Water 8,5 µl8,5 µl833 µl
Total22,5µl22,5µl 2205µl

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

A	B	C
Mix 1 Reverse transcription	Vol. (1x)	96 samples (+2 = 98 to keep some extra due to pipetting issues)
Random Hexamers (50µM)	1µL	98µL
dNTPs mix (10mM each)	1µL	98µL
Total	2µL	194µL

A	B	C
Mix 2 Reverse Transcription	Vol. (1x)	96 samples (+2 = 98 to keep some extra due to pipetting issues)
5x SSIV Buffer	4µL	392µL
100mM DTT	1µL	98µL
RNaseOUT or RNase Inhibitor	1µL	98µL
SSIV Reverse Transcriptase	1µL	98µL
Total	7µL	686µL

A	B	C	D	E	F
Primer	Sequence	Tm	Concentratio n inside of the pool *	Volume of primer within the pool	POOL
DENV1SA_1_LEFT	TACGTGGACCGACAAGAACAGT	58.1ºC	0,0075uM	2,5µl	1
DENV1SA_1_RIGHT	ACTATCATRTGTGGCTCTCCCC	57.2ºC	0,0075uM	2,5µl	1
DENV1SA_3_LEFT	CACACGTGGGACTTGGTCTAGA	58.4ºC	0,01125uM	7,5µl	1
DENV1SA_3_RIGHT	ACACACAAAGTTCGCGTCTTGT	57.6ºC	0,01125uM	7,5µl	1
DENV1SA_5_LEFT	CCTCACATTGGACTGCTCACCT	59.1ºC	0,01125uM	7,5µl	1
DENV1SA_5_RIGHT	TGCACTARRACAGTTCCATGCT	56.6ºC	0,01125uM	7,5µl	1
DENV1SA_7_LEFT	CGAGGAGCACGAAGGATGGC	60.6ºC	0,0075uM	2,5µl	1
DENV1SA_7_RIGHT	ATGATGTTCTCAAGACGCGTGG	57.5ºC	0,0075uM	2,5µl	1
DENV1SA_9_LEFT	TGGGAAGTTGAGGACTAYGGGT	58.7ºC	0,015uM	5µl	1
DENV1SA_9_RIGHT	TGTRGTTCTGAGRGATGGACCTC	57.8ºC	0,015uM	5µl	1
DENV1SA_11_LEFT	GATGACTGGAACACTGGCTGTT	57.4ºC	0,030uM	10µl	1
DENV1SA_11_RIGHDENV1SA_11_RIGHT	CACCGGAAGCCATGTTGTTTTT	56.7ºC	0,030uM	10µl	1
DENV1SA_13_LEFT	AASAAGAAGCAGAACACTCCGG	57.3ºC	0,015uM	5µl	1
DENV1SA_13_RIGHT	ACTGGCCCAGCTTGGTTCCAG	62.4ºC	0,015uM	5µl	1
DENV1SA_15_LEFT	ATGGAGTGGTGACAACAAGTGG	57.4ºC	0,015uM	5µl	1
DENV1SA_15_RIGHT	GCTGGATCGGTAAARTGTGCTTC	57.4ºC	0,015uM	5µl	1
DENV1SA_17_LEFT	ACGGGTRATYCAAYTGAGCAGRA	58.5ºC	0,01125uM	7,5µl	1
DENV1SA_17_RIGHT	CCTCTTCTCATGAGCTCCACA	56.3ºC	0,01125uM	7,5µl	1
DENV1SA_19_LEFT	AGTGTCTCAGGTGACCTAATATTGGA	57ºC	0,0075uM	2,5µl	1
DENV1SA_19_RIGHT	RGCTGCCACTGTCAGTATCATG	57.5ºC	0,0075uM	2,5µl	1
DENV1SA_21_LEFT	YGCAAAYCAGGCWGCYATATTGAT	57.6ºC	0,015uM	5µl	1
DENV1SA_21_RIGHT	GATGTTTGCCATGGACACTGCT	58.2ºC	0,015uM	5µl	1
DENV1SA_23_LEFT	ACAACCAAACATGCAGTGTCGA	57.5ºC	0,015uM	5µl	1
DENV1SA_23_RIGHT	TTTCGCACTAGCATCCCTCCAT	58.5ºC	0,015uM	5µl	1
DENV1SA_25_LEFT	ACCTAGATATYATTGGCCAGAGGA	55.9ºC	0,030uM	10µl	1
DENV1SA_25_RIGHT	ACCTTTCGTCTTCCACTGCTTC	57.3ºC	0,030uM	10µl	1
DENV1SA_27_LEFT	TGGAAGGAGAAGGACTGCACAA	58.4ºC	0,030uM	10µl	1
DENV1SA_27_RIGHT	CACRCAATCATCTCCGCTRATT	55.5ºC	0,030uM	10µl	1
DENV1SA_29_LEFT	ATGGAGCCTGAGAGAAACTGCT	58.2ºC	0,030uM	10µl	1
DENV1SA_29_RIGHT	GCYCCTTCGGGATCACTCTCAT	59.7ºC	0,030uM	10µl	1
DENV1SA_2_LEFT	TGTTGAACATAATRAACAGGAGGAA AAGA	55.9ºC	0,01125uM	7,5µl	2
DENV1SA_2_RIGHT	GAATCCTGGGTGTCKCAAAGCC	59.5ºC	0,01125uM	7,5µl	2
DENV1SA_4_LEFT	ACTGTGCATTGAAGCTAAAATATCAA ACA	56ºC	0,015uM	5µl	2
DENV1SA_4_RIGHT	ACCATTGTTTGTGGACAAGCCA	57.7ºC	0,015uM	5µl	2
DENV1SA_6_LEFT	AAACTGACYTTARAGGGGATGTCAT	56.1ºC	0,015uM	5µl	2
DENV1SA_6_RIGHT	ATATGCRGTCCCAAAAACCTGG	56.7ºC	0,015uM	5µl	2
DENV1SA_8_LEFT	AGGCTGACTCCCCAAAAAGACT	58.5ºC	0,015uM	5µl	2
DENV1SA_8_RIGHT	TTGATGGCAGCTGACATTAGCC	57.8ºC	0,015uM	5µl	2
DENV1SA_10_LEFT	GCAGGGCCATGGCACCTAGG	63.5ºC	0,0075uM	2,5µl	2
DENV1SA_10_RIGHT	TCCCCATCCTGTCTGAAGCATT	58.4ºC	0,0075uM	2,5µl	2
DENV1SA_12_LEFT	GGATTATGCATGGAARACAAYGGC	56.9ºC	0,030uM	10µl	2
DENV1SA_12_RIGHT	GTGAGTGTRTCATCCCTYTCTTCA	56.2ºC	0,030uM	10µl	2
DENV1SA_14_LEFT	AGGTCCCAAGTAGGAGTGGGAGT	61.2ºC	0,015uM	5µl	2
DENV1SA_14_RIGHT	CACCTCRTCCTCAATCTCTGGT	57.2ºC	0,015uM	5µl	2
DENV1SA_16_LEFT	GGGAGATAGTTGACCTCATGTGCCA	60.3ºC	0,015uM	5µl	2
DENV1SA_16_RIGHT	CCTGTCGGCCCGGAAATTTGC	61.7ºC	0,015uM	5µl	2
DENV1SA_18_LEFT	CAGAAGGGATCATCCCAGCCCT	60.9ºC	0,0075uM	2,5µl	2
DENV1SA_18_RIGHT	CCTCCTTGTTCGGAATTGTGCA	57.9ºC	0,0075uM	2,5µl	2
DENV1SA_20_LEFT	GCTGCTCATTCCAGARCCAGAC	59.2ºC	0,015uM	5µl	2
DENV1SA_20_RIGHT	ATGGGTTCACCTGGGAATAGCA	58.4ºC	0,015uM	5µl	2
DENV1SA_22_LEFT	TCCATCACACTGGCTACTGGAC	58.6ºC	0,015uM	5µl	2
DENV1SA_22_RIGHT	CCCACAACCGAGGTCTATGACT	58.4ºC	0,015uM	5µl	2
DENV1SA_24_LEFT	GCTYAGAGGAAACCAATTCTGCA	56.5ºC	0,030uM	10µl	2
DENV1SA_24_RIGHT	TGATCCTGATGGYTTGACCTCA	54.7ºC	0,030uM	10µl	2
DENV1SA_26_LEFT	CTGCACAAGAGAGGAGTTCACA	56.8ºC	0,015uM	5µl	2
DENV1SA_26_RIGHT	TATTCTTGTGTCCCATCCGGCT	58.3ºC	0,015uM	5µl	2
DENV1SA_28_LEFT	GAAACCCCCAAYCTAGCTRAGA	56.4ºC	0,030uM	10µl	2
DENV1SA_28_RIGHT	TAGCCGCTAGTCTCAGGTCTCT	58.8ºC	0,030uM	10µl	2
DENV1SA_30_LEFT	GGGCCACYAATATACAAGTAGCCA	57.6ºC	0,030uM	10µl	2
DENV1SA_30_RIGHT	CCCGCTGCTGCGTTATGTCT	60.4ºC	0,015uM	10µl	2
DENV1SA_31_RIGHT	CCTGTTGATTCAACAGCACCATTCCA	59.7ºC	0,015uM	10µl	2

A	B	C	D
Mix 1 Multiplex PCR	Vol. Pool 1 (1x)	Vol. Pool 2 (1x)	96 samples (+2) (pool1 or pool2)
Q5 Master Mix High fidelity 2X	12,5 µl	12,5 µl	1.225 µl
Pool primers (Pool1 ou Pool2) /Use concentration/	1,5 µl	1,5 µl	147 µl
Ultra Pure Water	8,5 µl	8,5 µl	833 µl
Total	22,5µl	22,5µl	2205µl

Public workspaceReverse transcription, primer pools preparation and multiplex PCR steps for DENV1 serotype for genomic sequencing

Reverse transcription, primer pools preparation and multiplex PCR steps for DENV1 serotype for genomic sequencing