TITLE: nCoV-2019 Illumina Miniseq sequencing protocol (2,000bp amplicon)
AUTHORS: Bruno Gomez-Gil, juli.encisoi

[DESCRIPTION]
This is a fork of the protocol https://dx.doi.org/10.17504/protocols.io.bh7hj9j6 but modified for tiled 2000bp  amplicons, tagmentation with Nextera XT, indexing, and sequencing with the Illumina Miniseq platform.
It has already produced very good sequences.
Much of this protocol is base on this paper: https://doi.org/10.1093/biomethods/bpaa014 

[GUIDELINES]
Tested with high viral copy numbers (<30 Ct).

[STEPS]
SECTION: cDNA preparation
2. We use the  
Mix (pipetting) the following components in Eppendorf tube.

Component                                                                Volume
       
Nuclease-free water                                               4 µL   
GoScriptTM Reaction Buffer, Random Primer   2 µL
GoScriptTM Enzyme Mix                                       0.4 µL
Final volume                                                            10 µL 
 

 


SECTION: cDNA preparation
3. Prepare the mastermix on ice, mix by pipetting.

Component                                                    Volume

GoScriptTM Reverse Transcription Mix     10 µL 
RNA                                                                  3 µL 
Final volume                                                   13 µL 


SECTION: cDNA preparation
4. Incubate the reaction as follows:

Anneal primers     25 °C for 5 min
Extension              42 °C  for 60 min 
Inactivation           70 °C  for 15 min 

Snap cool in a prechilled metal rack or on ice 1 min 
 

 

SECTION: Primer pool preparation
6. PRIMER STOCKS (100 micromolar (µM) )
If you have ordered each primer independently and need to generate primer pool stocks: add  5 µL of each primer from Pool 1 to a 1.5 mL Eppendorf labeled “Pool 1 (100µM)” and each primer from Pool 2 to a 1.5 mL Eppendorf labelled  “Pool 2 (100µM)”. These are your 100 micromolar (µM) stocks of each primer pool.


 

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

SECTION: Multiplex PCR
9. In the PCR hood set up the multiplex mastermix reaction as follows in 2 0.2mL PCR tubes. We use the
  

Component                                             Pool 1                Pool 2               Final Concentration

5X Kapa HotStart Buffer                       2.5 µL        2.5 µL            1 X      
25 mM MgCl2                                         0.75 µL     0.75 µL         1.5 millimolar (mM) 
10 mM dNTPs                                        0.25 µL      0.25 µL         0.2 millimolar (mM) 
Primer Pool 1 or 2 (10µM ea)              0.6 µL         0.6 µL           0.5 micromolar (µM) 
Kapa Taq HotStart Polymerase           0.1 µL         0.1 µL           0.5 U 
Nuclease-free water                              5.8 µL         5.8 µL

Final mastermix volume                      10.0 µL       10.0 µL

 

SECTION: Multiplex PCR
10. In the extraction and sample addition cabinet add 2.5 µL cDNA to each tube and mix well by pipetting.
The final volume will be 12.5 µL 
 


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

Step                                            Temperature     Time                    Cycles

Heat Activation                         98 °C        30 s    1
Denaturation                             95 °C        15 s     25-35 
Annealing and Extension        60 °C        5 min     25-35
Hold                                            4 °C            Indefinite              1
 


 

 



SECTION: Pooling and PCR quantification
14. Quantify DNA using a Qubit or other method.
Quantification using Nanodrop is not recommended for a good estimation of the final pool.             

 

SECTION: Pooling and PCR quantification
14.1. Protocol
 



SECTION: Normalisation
15. Label a  0.2 mL PCR tube for each sample.


SECTION: Normalisation
15.1. Adjust the amount of DNA in the tube to be 100 ng total per sample in 7.5 µL molecular grade water. 

 

SECTION: Sample preparation
1. Dilute the sample depending on the Ct values, this will reduce the likelihood of PCR-inhibition.

Ct range     Sample          Water

12-15         2 µL     198 µL 
16-18         2 µL     18 µL 
>18            no dilution

SECTION: Tagmentation
16.  WWe have reduced the amount of reagents used per reaction.

SECTION: Primer pool preparation
5. PRIMERS for this protocol are described in protocol nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon) and are the 2,000 bp option.
We selected for 2,000 bp amplicons so they could be more easily tagmented in the library preparation.
Two pooles are prepared, Pool1 with 18 primers and Pool2 with 16 primers.

 POOL1. Average Tm 61.01 °C 
          Name  Sequence  Direction  Minimum  Maximum  Length  Product Size  Tm  %GC  Hairpin Tm  Pair Dimer Tm  Self Dimer Tm    SARSCoV_2000_01_LEFT  ACCAACCAACTTTCGATCTCTTGT  forward  31  54  24  2049  60.7  41.7  None  None  None    SARSCoV_2000_01_RIGHT  ACACCACCTGTAATGTAGGCCA  reverse  2058  2079  22  2049  61.4  50  39  None  5    SARSCoV_2000_03_LEFT  TCGCACAAATGTCTACTTAGCTGT  forward  3772  3795  24  1814  60.6  41.7  None  0.7  None    SARSCoV_2000_03_RIGHT  GTGTGCCCATGTACATAACAGCT  reverse  5563  5585  23  1814  61.2  47.8  42.7  0.7  8.3    SARSCoV_2000_05_LEFT  CAATCATGCAATTGTTTTTCAGCTATTTTG  forward  7299  7328  30  1825  60.4  30  33  None  7.4    SARSCoV_2000_05_RIGHT  CGTGTGTCAGGGCGTAAACTTT  reverse  9102  9123  22  1825  61.6  50  None  None  None    SARSCoV_2000_07_LEFT  GGACGTACCATATTGGGTAGTGC  forward  10886  10908  23  1834  60.8  52.2  43.8  None  None    SARSCoV_2000_07_RIGHT  TCTGTCGTAGTGCAACAGGACT  reverse  12698  12719  22  1834  61.3  50  41.4  None  14    SARSCoV_2000_09_LEFT  ACCACTTCAGAGAGCTAGGTGT  forward  14477  14498  22  1925  60.5  50  None  None  None    SARSCoV_2000_09_RIGHT  ACAACCTGGAGCATTGCAAACA  reverse  16380  16401  22  1925  61.5  45.5  None  None  11.9    SARSCoV_2000_11_LEFT  TGGCATACCTAAGGACATGACCT  forward  18168  18190  23  1814  60.9  47.8  38.3  None  11.2    SARSCoV_2000_11_RIGHT  CAGTGAGTGGTGCACAAATCGT  reverse  19960  19981  22  1814  61.6  50  38.7  None  20.4    SARSCoV_2000_13_LEFT  TCCTCAGTTTTACATTCAACTCAGGA  forward  21695  21720  26  1937  60.2  38.5  45.2  None  0.9    SARSCoV_2000_13_RIGHT  TGACTAGCTACACTACGTGCCC  reverse  23610  23631  22  1937  61.5  54.5  37  None  None    SARSCoV_2000_15_LEFT  AGGAGTCAAATTACATTACACATAAACGAA  forward  25360  25389  30  1805  60.1  30  None  None  None    SARSCoV_2000_15_RIGHT  ACTGCTACTGGAATGGTCTGTGT  reverse  27142  27164  23  1805  61.6  47.8  None  None  None    SARSCoV_2000_17_LEFT  ACTTGTCACGCCTAAACGAACA  forward  27873  27894  22  1918  60.7  45.5  36.7  None  None    SARSCoV_2000_17_RIGHT  TAGGCAGCTCTCCCTAGCATTG  reverse  29769  29790  22  1918  61.6  54.5  45.3  None  None  
 
POOL2. Average Tm 61.05 °C 
 
          Name  Sequence  Direction  Minimum  Maximum  Length  Product Size  Tm  %GC  Hairpin Tm  Pair Dimer Tm  Self Dimer Tm    SARSCoV_2000_02_LEFT  AGGCCGCTATAACAATACTAGATGGA  forward  1956  1981  26  1923  61.3  42.3  None  None  None    SARSCoV_2000_02_RIGHT  CAGCGATCTTTTGTTCAACTTGCT  reverse  3855  3878  24  1923  60.8  41.7  None  None  None    SARSCoV_2000_04_LEFT  TCAACATGCCAATTTAGATTCTTGCA  forward  5473  5498  26  1929  60.3  34.6  None  8.1  None    SARSCoV_2000_04_RIGHT  GCTGAAATCGGGGCCATTTGTA  reverse  7380  7401  22  1929  61.5  50  None  8.1  4.4    SARSCoV_2000_06_LEFT  GCTGCTGAATGTACAATTTTTAAAGATGC  forward  9011  9039  29  2003  61.1  34.5  None  None  None    SARSCoV_2000_06_RIGHT  AACCAGTGGTGTGTACCCTTGA  reverse  10992  11013  22  2003  61.5  50  47  None  17.6    SARSCoV_2000_08_LEFT  TCACCTAATTTAGCATGGCCTCTT  forward  12620  12643  24  1956  60.1  41.7  None  None  3.3    SARSCoV_2000_08_RIGHT  CAGGGTCAGCAGCATACACAAG  reverse  14554  14575  22  1956  61.5  54.5  None  None  None    SARSCoV_2000_10_LEFT  TGCATACGTAGACCATTCTTATGTTGT  forward  16291  16317  27  1985  60.8  37  32.8  None  0.1    SARSCoV_2000_10_RIGHT  GCTTCTTCGCGGGTGATAAACA  reverse  18254  18275  22  1985  61.5  50  None  None  6.1    SARSCoV_2000_12_LEFT  GGACTACAAAAGAGATGCTCCAGC  forward  19878  19901  24  1920  61.5  50  42.5  11.2  None    SARSCoV_2000_12_RIGHT  ACCTCTTAGTACCATTGGTCCCA  reverse  21775  21797  23  1920  60.5  47.8  37.1  11.2  12.7    SARSCoV_2000_14_LEFT  GCTGAACATGTCAACAACTCATATGA  forward  23519  23544  26  1973  60.1  38.5  35.2  None  5.1    SARSCoV_2000_14_RIGHT  TGCAGTAGCGCGAACAAAATCT  reverse  25470  25491  22  1973  61.4  45.5  46.2  None  12.1    SARSCoV_2000_16_LEFT  TCTTATTACAAATTGGGAGCTTCGCA  forward  27051  27076  26  1995  61.3  38.5  37.6  None  None    SARSCoV_2000_16_RIGHT  GCTTCTTAGAAGCCTCAGCAGC  reverse  29024  29045  22  1995  61.6  54.5  43.6  None  30.4  
 

SECTION: Pooling and PCR quantification
13. Amplicon quantification to make an equimolar mixture.


 

SECTION: Pooling and PCR quantification
13.1. Put 1 µL  of each pool in a Nanodrop or similar spectometer and quantify the DNA concentration.
SECTION: Pooling and PCR quantification
13.2. Label a  1.5 mLEppendorf tube for each sample and make a equimolar mix with the two pools.
Calculate to achieve a final concentration of 50 1537

SECTION: Tagmentation
16.1. Label a  1.5 mLEppendorf tube for each sample and add in this order:
Component                            Volume

Tagment DNA buffer (TD)    3.75 µL 
cDNA 2 ng/µl                 3 µL 
           Pipette 10 times to mix

SECTION: Tagmentation
16.3. Centrifuge at 280 x g, 1 min, 20 °C 

SECTION: Tagmentation
16.4. Incubate in thermal cycler
55 °C  for 10 min 
10 °C 
 
SECTION: Tagmentation
16.5. Add 2 µL  of Neutralize Tagment Buffer (NT).
Pipette 10 times to mix
SECTION: Tagmentation
16.6. Centrifuge at 280 x g, 1 min, 20 °C 

SECTION: Tagmentation
16.7. Incubate at Room temperature  for 5 min °C 

SECTION: Tagmentation
16.2. Add Amplicon Tagment Mix 0.5 µL and pipette 10 times to mix
SECTION: Indexing
17. La siguiente reacción requiere de la enzima polimerasa 2x Ampigene HS Taq Mix Catalog # ENZ-NUC101-0200 y de los sets de Nextera XT. Cada set contiene 96 combinaciones de TAGs con esto podemos alcanzar 384 muestras usando los Nextera XT Index Kit v2 (Sets A,B,C y D) Catalog  # 20027213;20027214;20027215;20027216. 



SECTION: Tagmentation
16.8. Preserve the samples at 4-8 °C  until use.

SECTION: Primer pool preparation
8. POOLING OF PRIMERS
Label two 1.5 mL Eppendorf tubes, one as POOL1 and the another as POOL2.


SECTION: Primer pool preparation
8.1. Add 10 µL of each of the primers of set 1   to the Eppendorf tube labelled POOL1, final concentration will be 10 micromolar (µM)

SECTION: Primer pool preparation
8.2. Add 10 µL of each of the primers of set 2   to the Eppendorf tube labelled POOL2, final concentration will be 10 micromolar (µM) 

18. 
SECTION: Indexing
17.1. A cada tubo de reaccion agregar:
Component                                                                Volume
       
Nuclease-free water                                               10 µL   
2x Ampigene HS Taq Mix.                                      10 µL
Nextera XT index i5                                                1 µL
Nextera XT index i7                                                1 µL
ADN Tagmentado                                                   3 µL
Final volume                                                            25 µL 
SECTION: Indexing
17.2. Centrifuge at 280 x g, 1 min, 20 °C 
SECTION: Indexing
17.3. Set-up the following program on the thermal cycler:

Cover 100 °C

Step                                            Temperature     Time                    Cycles

Heat Activation                         72 °C        3 min    1
Denaturation                             95 °C        30 s     1
                                                  95 °C        10 s     
Annealing and Extension        55 °C        30 s     14
                                                  72 °C        30 s  
Hold                                            4 °C            Indefinite              1
SECTION: Purificación final y pooling
19. Al volumen que se tiene en el tubo agregar 0.8X de perlas magnéticas Ampure XP.

SECTION: Purificación final y pooling
19.1. Mezclar y spin-down.

SECTION: Purificación final y pooling
19.2. Llevar al magneto hasta formar el pellet y desechar el sobrenadante. 

SECTION: Purificación final y pooling
19.3. Agregar 150 uL de etanol 80% en posición contraria al pellet. Esperar 30 segundos.
Desechar el etanol.

SECTION: Purificación final y pooling
19.4. Retirar el exceso de etanol y secar las perlas por 5 min.

SECTION: Purificación final y pooling
19.5. Resuspender las perlas en 26 uL de Agua libre de nucleasas.
Mix y spin down. Incubar por 5 min a TA.

SECTION: Purificación final y pooling
19.6. Llevar al magneto hasta formar el pellet y transferir 25 uL del sobrenadante a un nuevo tubo previamente etiquetado.

SECTION: Purificación final y pooling
19.7. Cuantificar 2 uL por Qubit HS y analizar los tamaños mediante electroforesis en gel de agarosa al 1.0%.
SECTION: Secuenciacion de bibliotecas en plataforma IIlumina Miniseq
20. A partir de la concentración en ng/uL determinada por Qubit y obtenido el tamaño aproximado del fragmento, llevar cada una de las librerias a una concetracion de 4 nM.
Transferir 5 uL de cada librerías a 4 nM aun tubo previamente etiquetado para obtener el pool final.
Seguir el protocolo Library Denaturing and miniseq Sample Loading del kit