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

Laís Ceschini; Luisa Maria Inácio da Silva; Gabriel Luz Wallau

Aug 11, 2023

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

DOI

dx.doi.org/10.17504/protocols.io.5qpvo3rm7v4o/v1

¹Entomology department, Instituto Aggeu Magalhães (IAM), FIOCRUZ

Luisa Maria Inácio da Silva

DOI: dx.doi.org/10.17504/protocols.io.5qpvo3rm7v4o/v1

Protocol Citation: Laís Ceschini, Luisa Maria Inácio da Silva, Gabriel Luz Wallau 2023. Reverse transcription, primer pools preparation and multiplex PCR steps for CHIKV serotype genomic sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3rm7v4o/v1

Manuscript citation:

Machado, Lais Ceschini, et al. “Genome Sequencing Reveals Coinfection by Multiple Chikungunya Virus Genotypes in a Recent Outbreak in Brazil.” PLOS Neglected Tropical Diseases, edited by Fabrice Simon, vol. 13, no. 5, May 2019, p. e0007332, doi:https://doi.org/10.1371/journal.pntd.0007332.

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: 86340

Funders Acknowledgement:

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 CHIKV serotype strains.

Materials

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)1µL98µL
dNTPs mix (10mM each)1µL98µL
Total 2 µL194µL

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 Transcription
Vol.
(1x)96 samples (+2 = 98 to keep
some extra due to pipetting
issues)
5x SSIV Buffer4µL392µL
100mM DTT 1µL98µL
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 469µl and pool 2 of 460µl.

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


ABCDE
PrimerSequenceConcentration inside of
the pool *
Volume of
primer within
the poolPool
400_1_LEFT_1TGACACACGTAGCCTACCAGTT0,015uM10ul1
400_1_RIGHT_1CGCATCGGGCAAACGCAGTGGTA0,015uM 10ul1
400_3_LEFT_3GCAGACGTCGCGATATACCAAG0,015uM 10ul1
400_3_RIGHT_3CCAGCTCTTAAGTAGCATGCGG0,015uM 10ul1
400_5_LEFT_0GATGTGCAAGACTACCGACACG0,015uM 10ul1
400_5_RIGHT_0GACTGGGTATCAGGCCTCTTGT0,015uM 10ul1
400_7_LEFT_4CAAGAAGCCCAGGATGCTGAAA0,015uM 10ul1
400_7_RIGHT_4GCTATGCGTACACGTCTTCACT0,015uM 10ul1
400_9_LEFT_4GCAGAGAGGACAGAACACGAGT0,015uM 10ul1
400_9_RIGHT_4CTCTCTGTCTCATCACGTCGGT0,015uM 10ul1
400_11_LEFT_0AGCAGTGCGGCTTCTTCAATAT0,015uM 10ul1
400_11_RIGHT_0TGCCTAACTGCGTAAACTCCTTT0,015uM 10ul1
400_13_LEFT_2ATTAAGGAGTGGGAGGTGGAGC0,015uM 10ul1
400_13_RIGHT_2TCTAGAATGGACGCTGCCTCAG0,015uM 10ul1
400_15_LEFT_4GGGGAAAGAATGGAATGGCTGG0,015uM 10ul1
400_15_RIGHT_4CGTTCACTGGTTCTATCTGCGT0,015uM 10ul1
400_17_LEFT_0AACTGAACGCAGCCTTTGTAGG0,015uM 10ul1
400_17_RIGHT_0ACACCTGTGGAGAGGAGAGGTA0,015uM 10ul1
400_19_LEFT_1CATACAGATGCGGACCCAAGTG0,015uM 10ul1
400_19_RIGHT_1GTTCAGGAGTCATGGCATAACGG0,015uM 10ul1
400_21_LEFT_2GCGCGTAAGTCCAAGGGAATAC0,015uM 10ul1
400_21_RIGHT_2GTCTCCGCTGTTTCTTGTACGG0,015uM 10ul1
400_23_LEFT_1ACTTTCGGAGACTTCCTACCCG0,015uM 10ul1
400_23_RIGHT_1ACAGCCTCTCTTTAGTCTCTGGA0,015uM 10ul1
400_25_LEFT_2ACCAAATCACCGATGAGTATGATGC0,015uM 10ul1
400_25_RIGHT_2TCGTTATCCTGATAGGGCTGGC0,015uM 10ul1
400_27_LEFT_4AGGCCTAAGGTGCAGGTTATACA0,015uM 10ul1
400_27_RIGHT_4GCAGGTGACAGCTGGAAATCTC0,015uM 10ul1
400_29_LEFT_0CGATGAATTGATGGCAGCCAGA0,015uM 10ul1
400_29_RIGHT_0GCAAAGGTGGCCATGGACATTA0,015uM 10ul1
400_31_LEFT_1TTCTACAATAGGAGGTACCAGCCT0,015uM 10ul1
400_31_RIGHT_1TTCATGCACATTCTCTCTCTGCG0,015uM 10ul1
400_33_LEFT_3GATACCCGTGCACATGAAGTCC0,015uM 10ul1
400_33_RIGHT_3TTTTTCGTAGCAGCAGGGTGTG0,015uM 10ul1
400_35_LEFT_0CCACAAGACCGTACCTAGCTCA0,015uM 10ul1
400_35_RIGHT_0TGGTGAAATGGGTGCGTACATG0,015uM 10ul1
400_37_LEFT_3AATGTCACAACAGTCCGGCAAT0,015uM 10ul1
400_37_RIGHT_3TTGGGTGGTCAGGATACAGCAA0,015uM 10ul1
400_39_LEFT_1GGCCACCCGCATGAGATAATTC0,015uM 10ul1
400_39_RIGHT_1ATAGGACAATCAGGGCTGCCAG0,015uM 10ul1
400_41_LEFT_0CTTGGAACCAACGCTATCGCTT0,015uM 10ul1
400_41_RIGHT_0 AGCAGCCACAGTGATATTATTTCCT0,015uM 10ul1
400_43_LEFT_0ACCAGGACAATTTGGCGACATC0,015uM 10ul1
400_43_RIGHT_0ATACCTCACACGACATGTCCGT0,015uM 10ul1
400_45_LEFT_3CTACACAAGTACACTGTGCAGCC0,015uM 10ul1
400_45_RIGHT_3TGTTATTCAGGGGTTGTTCAGCC0,015uM 10ul1
400_2_LEFT_0CCAGCAAGGAGGATGATGTCGGAC0,015uM 10ul2
400_2_RIGHT_0TGTGTCGAACCCTACCCAGTAC0,015uM 10ul2
400_4_LEFT_0TGTTCTCAGTAGGGTCAACGCT0,015uM 10ul2
400_4_RIGHT_0GGATGCCGGTCATTTGATCACA0,015uM 10ul2
400_6_LEFT_1TGAGAAGCTTTTGGGGGTCAGA0,015uM 10ul2
400_6_RIGHT_1ACATCTTCCTGTGCTGCCTGTA0,015uM 10ul2
400_8_LEFT_0ACTTTCCCCGCAGACCGTATTA0,015uM 10ul2
400_8_RIGHT_0CAGCTTCTTCCTTCTTGCAGCA0,015uM 10ul2
400_10_LEFT_0ACCTGGTGACTAGCGGAAAGAA0,015uM 10ul2
400_10_RIGHT_0GACGACACAATGGCAGTCACAG0,015uM 10ul2
400_12_LEFT_0GAGGGTGGGTTAAACAACTGCA0,015uM 10ul2
400_12_RIGHT_0TTATCCCCGCTGTTTCGAGGAT0,015uM 10ul2
400_14_LEFT_1ACGCGGATAACCACTGGGATAA0,015uM 10ul2
400_14_RIGHT_1TTATAGCCGCTAACCAGGAGCA0,015uM 10ul2
400_16_LEFT_0AGGTGACTCACTGAGACTGCTC0,015uM 10ul2
400_16_RIGHT_0ATCGTTCTTCGCGATGTCCATG0,015uM 10ul2
400_18_LEFT_2GGACCAAACTTCTCAAATTACACGGA0,015uM 10ul2
400_18_RIGHT_2CCAAACTACTGTCAGGGTGCAC0,015uM 10ul2
400_20_LEFT_4CAGAAATGCCCGGTGGATGATG0,015uM 10ul2
400_20_RIGHT_4ATCGGCGCTTAGATCAAACTGAC0,015uM 10ul2
400_22_LEFT_0GAGGGAGAAACCTGACCGTGAT0,015uM 10ul2
400_22_RIGHT_0AGTCATAACTCGTCGTCCGTGT0,015uM 10ul2
400_24_LEFT_4CACGGCCAATAGAAGCAGGTATC0,015uM 10ul2
400_24_RIGHT_4TTGACGGATTGAATGTCGCTCG0,015uM 10ul2
400_26_LEFT_0ACCCACTTTGGACTCAGCAGTA0,015uM 10ul2
400_26_RIGHT_0AGGACGGCGTTCAATCTCCTAA0,015uM 10ul2
400_28_LEFT_0CCAGGATGATTCACTTGCGCTT0,015uM 10ul2
400_28_RIGHT_0GGAGCTTTCTGGGATACAACTGC0,015uM 10ul2
400_30_LEFT_1GATGGCAACGAACAGGGCTAAT0,015uM 10ul2
400_30_RIGHT_1GGTCTGGGTCTGATGACTTGGA0,015uM 10ul2
400_32_LEFT_3CCCCCAAAAAGAAACCGGTTCA0,015uM 10ul2
400_32_RIGHT_3GAGTACTGTACTGCTCCGTGGT0,015uM 10ul2
400_34_LEFT_0CGTCACGAAAATCACCCCTGAG0,015uM 10ul2
400_34_RIGHT_0TCTGTCGCTTCGTTTCTGATGC0,015uM 10ul2
400_36_LEFT_0CCGTGCACGATTACTGGAACAA0,015uM 10ul2
400_36_RIGHT_0CACAATTGCACTTGTACCGCAC0,015uM 10ul2
400_38_LEFT_1TCCTCTGGCAAATGTGACATGC0,015uM 10ul2
400_38_RIGHT_1CACCCACCATCGACAGGAGTAT0,015uM 10ul2
400_40_LEFT_1TATACCTGTGGAACGAGCAGCA0,015uM 10ul2
400_40_RIGHT_1TGTACCGCAGCATTTCACGTAC0,015uM 10ul2
400_42_LEFT_4TCAGCATACAGGGCTCATACCG0,015uM 10ul2
400_42_RIGHT_4GACGGTCTCTGCAGTACCAGTT0,015uM 10ul2
400_44_LEFT_0ATCTCCATCGACATACCGGACG0,015uM 10ul2
400_44_RIGHT_0TGTGACGCCGGGTAATTGACTA0,015uM 10ul2
400_46_LEFT_1TCCCTAAAGAGACACACCGCAT0,015uM 10ul2
400_46_RIGHT_1TCTTAGCTATATATGGTGTGTCTCTTAG
GG 0,015uM 10ul2
*approximate concentration of each primer in the 25µl PCR reaction.

Note: The primers were designed using the https://primalscheme.com based on the
KP164576, KP164571, KP164572, KP164568, KP164570 and KP164569 reference genomes (Machado, 2019).

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 PCRVol. Pool 1
(1x)Vol. Pool 2
(1x)96 samples (+2) (pool1 ou pool2)
Q5 Master Mix High
fidelity 2X
12,5 µl12,5 µl1.225 µl
Pool primers (Pool1 or
Pool2) /Use concentration1,7 µl 1,7 µl 166,6 µl
Ultra Pure Water 8,3 µl 8,3 µl 813,3 µl
Total22,5µl 22,5µl 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	2 µL	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
Primer	Sequence	Concentration inside of the pool *	Volume of primer within the pool	Pool
400_1_LEFT_1	TGACACACGTAGCCTACCAGTT	0,015uM	10ul	1
400_1_RIGHT_1	CGCATCGGGCAAACGCAGTGGTA	0,015uM	10ul	1
400_3_LEFT_3	GCAGACGTCGCGATATACCAAG	0,015uM	10ul	1
400_3_RIGHT_3	CCAGCTCTTAAGTAGCATGCGG	0,015uM	10ul	1
400_5_LEFT_0	GATGTGCAAGACTACCGACACG	0,015uM	10ul	1
400_5_RIGHT_0	GACTGGGTATCAGGCCTCTTGT	0,015uM	10ul	1
400_7_LEFT_4	CAAGAAGCCCAGGATGCTGAAA	0,015uM	10ul	1
400_7_RIGHT_4	GCTATGCGTACACGTCTTCACT	0,015uM	10ul	1
400_9_LEFT_4	GCAGAGAGGACAGAACACGAGT	0,015uM	10ul	1
400_9_RIGHT_4	CTCTCTGTCTCATCACGTCGGT	0,015uM	10ul	1
400_11_LEFT_0	AGCAGTGCGGCTTCTTCAATAT	0,015uM	10ul	1
400_11_RIGHT_0	TGCCTAACTGCGTAAACTCCTTT	0,015uM	10ul	1
400_13_LEFT_2	ATTAAGGAGTGGGAGGTGGAGC	0,015uM	10ul	1
400_13_RIGHT_2	TCTAGAATGGACGCTGCCTCAG	0,015uM	10ul	1
400_15_LEFT_4	GGGGAAAGAATGGAATGGCTGG	0,015uM	10ul	1
400_15_RIGHT_4	CGTTCACTGGTTCTATCTGCGT	0,015uM	10ul	1
400_17_LEFT_0	AACTGAACGCAGCCTTTGTAGG	0,015uM	10ul	1
400_17_RIGHT_0	ACACCTGTGGAGAGGAGAGGTA	0,015uM	10ul	1
400_19_LEFT_1	CATACAGATGCGGACCCAAGTG	0,015uM	10ul	1
400_19_RIGHT_1	GTTCAGGAGTCATGGCATAACGG	0,015uM	10ul	1
400_21_LEFT_2	GCGCGTAAGTCCAAGGGAATAC	0,015uM	10ul	1
400_21_RIGHT_2	GTCTCCGCTGTTTCTTGTACGG	0,015uM	10ul	1
400_23_LEFT_1	ACTTTCGGAGACTTCCTACCCG	0,015uM	10ul	1
400_23_RIGHT_1	ACAGCCTCTCTTTAGTCTCTGGA	0,015uM	10ul	1
400_25_LEFT_2	ACCAAATCACCGATGAGTATGATGC	0,015uM	10ul	1
400_25_RIGHT_2	TCGTTATCCTGATAGGGCTGGC	0,015uM	10ul	1
400_27_LEFT_4	AGGCCTAAGGTGCAGGTTATACA	0,015uM	10ul	1
400_27_RIGHT_4	GCAGGTGACAGCTGGAAATCTC	0,015uM	10ul	1
400_29_LEFT_0	CGATGAATTGATGGCAGCCAGA	0,015uM	10ul	1
400_29_RIGHT_0	GCAAAGGTGGCCATGGACATTA	0,015uM	10ul	1
400_31_LEFT_1	TTCTACAATAGGAGGTACCAGCCT	0,015uM	10ul	1
400_31_RIGHT_1	TTCATGCACATTCTCTCTCTGCG	0,015uM	10ul	1
400_33_LEFT_3	GATACCCGTGCACATGAAGTCC	0,015uM	10ul	1
400_33_RIGHT_3	TTTTTCGTAGCAGCAGGGTGTG	0,015uM	10ul	1
400_35_LEFT_0	CCACAAGACCGTACCTAGCTCA	0,015uM	10ul	1
400_35_RIGHT_0	TGGTGAAATGGGTGCGTACATG	0,015uM	10ul	1
400_37_LEFT_3	AATGTCACAACAGTCCGGCAAT	0,015uM	10ul	1
400_37_RIGHT_3	TTGGGTGGTCAGGATACAGCAA	0,015uM	10ul	1
400_39_LEFT_1	GGCCACCCGCATGAGATAATTC	0,015uM	10ul	1
400_39_RIGHT_1	ATAGGACAATCAGGGCTGCCAG	0,015uM	10ul	1
400_41_LEFT_0	CTTGGAACCAACGCTATCGCTT	0,015uM	10ul	1
400_41_RIGHT_0	AGCAGCCACAGTGATATTATTTCCT	0,015uM	10ul	1
400_43_LEFT_0	ACCAGGACAATTTGGCGACATC	0,015uM	10ul	1
400_43_RIGHT_0	ATACCTCACACGACATGTCCGT	0,015uM	10ul	1
400_45_LEFT_3	CTACACAAGTACACTGTGCAGCC	0,015uM	10ul	1
400_45_RIGHT_3	TGTTATTCAGGGGTTGTTCAGCC	0,015uM	10ul	1
400_2_LEFT_0	CCAGCAAGGAGGATGATGTCGGAC	0,015uM	10ul	2
400_2_RIGHT_0	TGTGTCGAACCCTACCCAGTAC	0,015uM	10ul	2
400_4_LEFT_0	TGTTCTCAGTAGGGTCAACGCT	0,015uM	10ul	2
400_4_RIGHT_0	GGATGCCGGTCATTTGATCACA	0,015uM	10ul	2
400_6_LEFT_1	TGAGAAGCTTTTGGGGGTCAGA	0,015uM	10ul	2
400_6_RIGHT_1	ACATCTTCCTGTGCTGCCTGTA	0,015uM	10ul	2
400_8_LEFT_0	ACTTTCCCCGCAGACCGTATTA	0,015uM	10ul	2
400_8_RIGHT_0	CAGCTTCTTCCTTCTTGCAGCA	0,015uM	10ul	2
400_10_LEFT_0	ACCTGGTGACTAGCGGAAAGAA	0,015uM	10ul	2
400_10_RIGHT_0	GACGACACAATGGCAGTCACAG	0,015uM	10ul	2
400_12_LEFT_0	GAGGGTGGGTTAAACAACTGCA	0,015uM	10ul	2
400_12_RIGHT_0	TTATCCCCGCTGTTTCGAGGAT	0,015uM	10ul	2
400_14_LEFT_1	ACGCGGATAACCACTGGGATAA	0,015uM	10ul	2
400_14_RIGHT_1	TTATAGCCGCTAACCAGGAGCA	0,015uM	10ul	2
400_16_LEFT_0	AGGTGACTCACTGAGACTGCTC	0,015uM	10ul	2
400_16_RIGHT_0	ATCGTTCTTCGCGATGTCCATG	0,015uM	10ul	2
400_18_LEFT_2	GGACCAAACTTCTCAAATTACACGGA	0,015uM	10ul	2
400_18_RIGHT_2	CCAAACTACTGTCAGGGTGCAC	0,015uM	10ul	2
400_20_LEFT_4	CAGAAATGCCCGGTGGATGATG	0,015uM	10ul	2
400_20_RIGHT_4	ATCGGCGCTTAGATCAAACTGAC	0,015uM	10ul	2
400_22_LEFT_0	GAGGGAGAAACCTGACCGTGAT	0,015uM	10ul	2
400_22_RIGHT_0	AGTCATAACTCGTCGTCCGTGT	0,015uM	10ul	2
400_24_LEFT_4	CACGGCCAATAGAAGCAGGTATC	0,015uM	10ul	2
400_24_RIGHT_4	TTGACGGATTGAATGTCGCTCG	0,015uM	10ul	2
400_26_LEFT_0	ACCCACTTTGGACTCAGCAGTA	0,015uM	10ul	2
400_26_RIGHT_0	AGGACGGCGTTCAATCTCCTAA	0,015uM	10ul	2
400_28_LEFT_0	CCAGGATGATTCACTTGCGCTT	0,015uM	10ul	2
400_28_RIGHT_0	GGAGCTTTCTGGGATACAACTGC	0,015uM	10ul	2
400_30_LEFT_1	GATGGCAACGAACAGGGCTAAT	0,015uM	10ul	2
400_30_RIGHT_1	GGTCTGGGTCTGATGACTTGGA	0,015uM	10ul	2
400_32_LEFT_3	CCCCCAAAAAGAAACCGGTTCA	0,015uM	10ul	2
400_32_RIGHT_3	GAGTACTGTACTGCTCCGTGGT	0,015uM	10ul	2
400_34_LEFT_0	CGTCACGAAAATCACCCCTGAG	0,015uM	10ul	2
400_34_RIGHT_0	TCTGTCGCTTCGTTTCTGATGC	0,015uM	10ul	2
400_36_LEFT_0	CCGTGCACGATTACTGGAACAA	0,015uM	10ul	2
400_36_RIGHT_0	CACAATTGCACTTGTACCGCAC	0,015uM	10ul	2
400_38_LEFT_1	TCCTCTGGCAAATGTGACATGC	0,015uM	10ul	2
400_38_RIGHT_1	CACCCACCATCGACAGGAGTAT	0,015uM	10ul	2
400_40_LEFT_1	TATACCTGTGGAACGAGCAGCA	0,015uM	10ul	2
400_40_RIGHT_1	TGTACCGCAGCATTTCACGTAC	0,015uM	10ul	2
400_42_LEFT_4	TCAGCATACAGGGCTCATACCG	0,015uM	10ul	2
400_42_RIGHT_4	GACGGTCTCTGCAGTACCAGTT	0,015uM	10ul	2
400_44_LEFT_0	ATCTCCATCGACATACCGGACG	0,015uM	10ul	2
400_44_RIGHT_0	TGTGACGCCGGGTAATTGACTA	0,015uM	10ul	2
400_46_LEFT_1	TCCCTAAAGAGACACACCGCAT	0,015uM	10ul	2
400_46_RIGHT_1	TCTTAGCTATATATGGTGTGTCTCTTAG GG	0,015uM	10ul	2

A	B	C	D
Mix 1 Multiplex PCR	Vol. Pool 1 (1x)	Vol. Pool 2 (1x)	96 samples (+2) (pool1 ou pool2)
Q5 Master Mix High fidelity 2X	12,5 µl	12,5 µl	1.225 µl
Pool primers (Pool1 or Pool2) /Use concentration	1,7 µl	1,7 µl	166,6 µl
Ultra Pure Water	8,3 µl	8,3 µl	813,3 µl
Total	22,5µl	22,5µl	2205µl

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

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