TITLE: Protocol for SCV-2000bp: a primer panel for SARS-CoV-2 full-genome sequencing.v3.Neb
AUTHORS: Valeriia Kaptelova, Speranskaya AS, et.al

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here we present a new primer panel that allows amplifying the complete genome of SARS-CoV-2 (the causative virus of COVID-19) using 17 primer pairs (in four pools). Our results demonstrate that our method allows producing full genomes when we use RNA extract from SARS-CoV-2 positive clinical samples which have a cycle threshold (Ct) in the range of 13 to 26. The resulting primer set exhibits the coverage of the entire viral genome except for only 8 bp on 5'- and 80 bp on 3'- ends in comparison with the reference genome in GenBank (accession number MT121215.1).</div></div>

[STEPS]
?. [1. cDNA]
RNA samples should be stored at -80 °C and thawed on ice.NGS library preparation and sequencing:cDNAReverse transcription reaction was performed using 10 μL of the RNA samples, random hexanucleotide primers, and Reverta-L kit (AmpliSens, Russia) according to the manufacturer’s instructions. Prepare a ready-to-use reagent mix for 12 reactions.1.1. Add 5 µl of RT-G-mix-1 to the tube containing RT-mix, 1.2. Add 6 µl of Revertase (MMlv) into the tube with reagent mix, 1.3. Mix well1.4. Dispense 10 µl of ready-to-use reagent mix into each prepared test tube (0.2 ml ).1.5. Add 10 µl RNA-sample to the appropriate test tube with a ready-to-use reagent mix. Place the test tubes into the thermocycler and incubate at 37 °С for 30 minutes.1.6. Dilute each cDNA sample in the ratio 1:1 with DNA-buffer. To do that, add 20 µl DNAbuffer to each test tube. Carefully mix, using the pipette (10 times).
?. [2. Amplification]
The cDNA was immediately used as a template for the amplification of genome fragments.The primer panel you can find here: see Table 1.Table 1. Primers for whole-genome sequencing of the SARS-CoV-2.  PrimerName    PrimerSequence 5’-3’    Start*    End*    Overlap    Pool    ProductLength    CV_1_F    gtttataccttcccaggtaacaaacc    8    33    155    5    2054    CV_1_R    actgaacaacaccacctgtaatgtag    2087    2062    CV_2_F    ttctcccgcactcttgaaactg    1907    1928    134    3    1808    CV_2_R    ggtcagcaccaaaaataccagct    3737    3715    CV_3_F    agcggacacaatcttgctaaaca    3581    3603    132    4    1946    CV_3_R    ggttgtctgctgttgtccacaa    5548    5527    CV_4_F    ttgtgcacttatcttagcctactgt    5395    5419    141    5    1839    CV_4_R    tgccaaaaaccactctgcaact    7255    7234    CV_5_F    cactattgcaacctactgtactggt    7093    7117    126    2    2009    CV_5_R    cgtgtgtcagggcgtaaacttt    9123    9102    CV_6_F    gtacactgactttgcaacatcagc    8977    9000    197    3    1851    CV_6_R    gcacacatatctaaaacggcaattc    10851    10827    CV_7_F    agcagctggtacggacacaac    10630    10650    144    1    2028    CV_7_R    tttgacagcagaattggccctt    12679    12658    CV_8_F    gtgtgatggtacaacatttacttatgc    12514    12540    130    2    1793    CV_8_R    tgggtggtatgtctgatcccaa    14328    14307    CV_9_F    ccttgaccagggctttaactgc    14177    14198    132    1    1783    CV_9_R    ttacgatatcatctacaaaacagccg    15985    15960    CV_10_F    agcaaaatgttggactgagactga    15828    15851    162    3    1906    CV_10_R    ccaagcagggttacgtgtaagg    17754    17733    CV_11_F    gctgaaattgttgacactgtgagt    17572    17595    188    1    1892    CV_11_R    agcaccacctaaattgcaacgt    19485    19464    CV_12_F    ggttgtgatggtggcagtttgt    19276    19297    74    2    1944    CV_12_R    taacaaaggctgtccaccatgc    21241    21220    CV_13_F    ttggaggttccgtggctataaaga    21146    21169    28    4    1733    CV_13_R    ccaccaaccttagaatcaagattg    22902    22879    CV_14_F    caggctgcgttatagcttggaa    22851    22872    242    2    1999    CV_14_R    aaccagtgtgtgccatttgaa    24870    24850    CV_15_F    gcagaaatcagagcttctgctaatc    24608    24632    237    3    1959    CV_15_R    actaggttccattgttcaaggagc    26590    26567    CV_16_F    ttacactagccatccttactgcg    26330    26352    457    1    2000    CV_16_R    actgccagttgaatctgagggt    28351    28330    CV_17_F    acttgtcacgcctaaacgaaca    27873    27894    -    5    1896    CV_17_R    taggcagctctccctagcattg    29790    29769  *Comparing to the reference genome in GenBank, accession MT121215.1  Five multiplexed primer pools each sample    cDNA         pM    μl    μl    pool
   1    12,5    0,5    5    pool
  2    12,5    0,4    5    pool
  3    12.5    0,4    5    pool
  4    10    0,6    5    pool 5    16    0,35    5  Set up the following four PCR master mixes, one for each of the four multiplexed primer pools (multiply below volumes by number of reactions plus desired overage):Q5 High-Fidelity DNA Polymerase was used according to the manufacturer’s instructions (New England BioLabs, NEB).  Component
      Volume
  per reaction (μl)    Q5 master mix    12,5    primer
  pool (1-5<b style="mso-bidi-font-weight:
  normal">)*    variable (0,35-0,6) *    H2O    Variable to 25    cDNA    5    Total
  volume:    25  Amplify samples using the following PCR conditions:  °C    Time    Cycles    98    0:30 s    1    98    0:10 s    35    64    0:30 s    72    2:30    72    3:00    1
PrimerName    PrimerSequence 5’-3’    Start*    End*    Overlap    Pool    ProductLength    CV_1_F    gtttataccttcccaggtaacaaacc    8    33    155    5    2054    CV_1_R    actgaacaacaccacctgtaatgtag    2087    2062    CV_2_F    ttctcccgcactcttgaaactg    1907    1928    134    3    1808    CV_2_R    ggtcagcaccaaaaataccagct    3737    3715    CV_3_F    agcggacacaatcttgctaaaca    3581    3603    132    4    1946    CV_3_R    ggttgtctgctgttgtccacaa    5548    5527    CV_4_F    ttgtgcacttatcttagcctactgt    5395    5419    141    5    1839    CV_4_R    tgccaaaaaccactctgcaact    7255    7234    CV_5_F    cactattgcaacctactgtactggt    7093    7117    126    2    2009    CV_5_R    cgtgtgtcagggcgtaaacttt    9123    9102    CV_6_F    gtacactgactttgcaacatcagc    8977    9000    197    3    1851    CV_6_R    gcacacatatctaaaacggcaattc    10851    10827    CV_7_F    agcagctggtacggacacaac    10630    10650    144    1    2028    CV_7_R    tttgacagcagaattggccctt    12679    12658    CV_8_F    gtgtgatggtacaacatttacttatgc    12514    12540    130    2    1793    CV_8_R    tgggtggtatgtctgatcccaa    14328    14307    CV_9_F    ccttgaccagggctttaactgc    14177    14198    132    1    1783    CV_9_R    ttacgatatcatctacaaaacagccg    15985    15960    CV_10_F    agcaaaatgttggactgagactga    15828    15851    162    3    1906    CV_10_R    ccaagcagggttacgtgtaagg    17754    17733    CV_11_F    gctgaaattgttgacactgtgagt    17572    17595    188    1    1892    CV_11_R    agcaccacctaaattgcaacgt    19485    19464    CV_12_F    ggttgtgatggtggcagtttgt    19276    19297    74    2    1944    CV_12_R    taacaaaggctgtccaccatgc    21241    21220    CV_13_F    ttggaggttccgtggctataaaga    21146    21169    28    4    1733    CV_13_R    ccaccaaccttagaatcaagattg    22902    22879    CV_14_F    caggctgcgttatagcttggaa    22851    22872    242    2    1999    CV_14_R    aaccagtgtgtgccatttgaa    24870    24850    CV_15_F    gcagaaatcagagcttctgctaatc    24608    24632    237    3    1959    CV_15_R    actaggttccattgttcaaggagc    26590    26567    CV_16_F    ttacactagccatccttactgcg    26330    26352    457    1    2000    CV_16_R    actgccagttgaatctgagggt    28351    28330    CV_17_F    acttgtcacgcctaaacgaaca    27873    27894    -    5    1896    CV_17_R    taggcagctctccctagcattg    29790    29769
Five multiplexed primer pools each sample    cDNA         pM    μl    μl    pool
   1    12,5    0,5    5    pool
  2    12,5    0,4    5    pool
  3    12.5    0,4    5    pool
  4    10    0,6    5    pool 5    16    0,35    5
Component
      Volume
  per reaction (μl)    Q5 master mix    12,5    primer
  pool (1-5<b style="mso-bidi-font-weight:
  normal">)*    variable (0,35-0,6) *    H2O    Variable to 25    cDNA    5    Total
  volume:    25
°C    Time    Cycles    98    0:30 s    1    98    0:10 s    35    64    0:30 s    72    2:30    72    3:00    1
[SAFE STOPPING POINT]
?. [3. 1,7% agarose electrophoresis.]
Then products of the amplification reaction were analyzed with electrophoresis using 1,7% agarose gel stained with SYBR Green. The samples in which visible PCR products of the expected size were found were used for PCR reactions with primer pools. Then products of PCR were visualized using 1,7% agarose electrophoresis stained with SYBR Green.
?. [4. mix]
Amplified fragments were mixed in equimolar amounts according to the visual estimation of concentration.
?. [5. Clean up]
Clean up (in the ratio 0,7x).Clean up PCR products of the expected size (1700-2100 bp) from the reaction mixture and to remove the nonspecific short fragments obtained during the amplification step.
?. [5. Clean up]
Premixed amplicons were cleaned in the ratio 0,7x using Agencourt AMPure XP (Beckman Coulter, Danvers, MA, USA)
?. [5. Clean up]
Incubate at Room temperature for
?. [5. Clean up]
Place the tubes on a magnet: allow beads to separate for
?. [5. Clean up]
Remove supernatant.
?. [5. Clean up]
Add 200 µl 80% fresh ethanol to wash the beads, incubate for
?. [5. Clean up]
Remove 200 µl 80% EtOH.
?. [5. Clean up]
Repeat steps 5.5-5.6.
?. [5. Clean up]
Briefly spin the tubes and remove additional ethanol. Ensure any visible quantities of ethanol are removed.
?. [5. Clean up]
Leave the beads in open tubes for
?. [5. Clean up]
Add low TE and incubate for .
?. [5. Clean up]
Place the tubes on a magnet: allow beads to separate for
?. [5. Clean up]
Transfer supernatant into the new tubes.SAFE STOPPING POINT!Store at -20 °C.
?. [6. Qubit]
Qubit dsDNA HS Assay Kit.Prepare the Qubit® working solution by diluting the Qubit® dsDNA HS Reagent 1:200 in Qubit® dsDNA HS Buffer. Use a clean plastic tube each time you prepare a Qubit® working solution. Add Qubit® working solution to individual assay tubes so that the final volume in each tube after adding a sample is 200 µL. Note: Your sample can be anywhere from 1–20 µL (We use 1µL). Add a corresponding volume of Qubit® working solution to each assay tube: anywhere from 180–199 µL.
?. [7.	Covaris.]
Covaris.PCR products (50 µl)were sheared in microTUBE-50 AFA Fiber Screw-Cap (PN 520166) using Covaris M220 (Covaris, Woburn, MA) using the following settings: peak incident power (W)- 75, duty factor- 5,5%, cycles per burst-200, treatment Time (s) - 52, temperature (°C)-20, sample volume (μl)- 50.
?. [8.	Clean up ]
Clean up (in the ratio 1,5x)See point 5.2-5.12
?. [9.	Qubit ]
Qubit dsDNA HS Assay Kit.See point 6.
?. [10. End repair  with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)]
End repair  with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)Starting Material: 11-100 ng in total reaction. Mix the following components in a sterile, nuclease-free tube:  AB1  COMPONENT VOLUME    VOLUME, µL, PER REACTION  2  (green) NEBNext
  Ultra II End Prep Enzyme Mix    3  3  &nbsp;(green) NEBNext Ultra II End Prep Reaction
  Buffer     7  4  Fragmented DNA (11-100 ng in total reaction)    variable (1-5 µL)  5  nuclease free water    variable (45- 49&nbsp; µL)  6  Total Volume    60  Set a 100 μl or 200 μl pipette to 50 μl and then gently pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program: for  for Hold atProceed directly to NEBNext Ultra II Ligation Module NEB E7595.
AB1  COMPONENT VOLUME    VOLUME, µL, PER REACTION  2  (green) NEBNext
  Ultra II End Prep Enzyme Mix    3  3  &nbsp;(green) NEBNext Ultra II End Prep Reaction
  Buffer     7  4  Fragmented DNA (11-100 ng in total reaction)    variable (1-5 µL)  5  nuclease free water    variable (45- 49&nbsp; µL)  6  Total Volume    60
20 °C
65 °C
4 °C
?. [11. Ligation with NEBNext Ultra II Ligation Module (E7595)]
Ligation with NEBNext Ultra II Ligation Module (E7595) and Y-shaped adapters compatible with Nextera XT Index Kit.need to dilute adaptor to 3 pM (for DNA input was 11-100 ng in total reaction) (0,6 pM for 1-10 ng in total reaction)Add the following components directly to the End Prep reaction mixture (60 µl) and mix well:  AB1  COMPONENT VOLUME    VOLUME, µL, PER REACTION  2  Y-shaped
  adapters compatible with Nextera XT Index Kit (dilution adapter)    2,5  3  &nbsp;(red) NEBNext Ligation Enhancer
      1  4  (red) NEBNext
  Ultra II Ligation Master Mix    30  5  Total Volume    93,5  Note: The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours at 4°C. We do not recommend premixing the Ligation Master Mix, Ligation Enhancer and adaptor prior to use in the Adaptor Ligation Step.Set a 100 µl or 200 µl pipette to 80 µl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube. (Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).Incubate at for  in a thermocycler with the heated lid off.
AB1  COMPONENT VOLUME    VOLUME, µL, PER REACTION  2  Y-shaped
  adapters compatible with Nextera XT Index Kit (dilution adapter)    2,5  3  &nbsp;(red) NEBNext Ligation Enhancer
      1  4  (red) NEBNext
  Ultra II Ligation Master Mix    30  5  Total Volume    93,5
20 °C
?. [12.	Clean up]
Clean up (in the ratio 1,5x)See point 5.2-5.12
?. [13.	Indexing PCR]
Indexing PCR  AB1  COMPONENT VOLUME    VOLUME, µL, PER REACTION  2  DNA    8,5  3  &nbsp;Q5 Master Mix     12,5  4  Index /i5 Primer (5pM)    2  5  Index&nbsp; /i7 Primer (5pM)    2  6  Total (mix)    25  Amplify samples using the following PCR conditions:  ABC1  °C    Time    Cycles  2  98    0:30 s    1  3  98    0:10 s    8  4  65 (detect)    1:15 m
AB1  COMPONENT VOLUME    VOLUME, µL, PER REACTION  2  DNA    8,5  3  &nbsp;Q5 Master Mix     12,5  4  Index /i5 Primer (5pM)    2  5  Index&nbsp; /i7 Primer (5pM)    2  6  Total (mix)    25
ABC1  °C    Time    Cycles  2  98    0:30 s    1  3  98    0:10 s    8  4  65 (detect)    1:15 m
?. [14. Clean up]
Clean up (in the ratio 1,2x)See point 5.2-5.12
?. [15.	Agilent 2100 Bioanalyzer.]
Agilent 2100 Bioanalyzer.The quality and fragment length distribution of the obtained libraries were evaluated with Agilent Bioanalyzer 2100 (Agilent Technologies, USA).
?. [16.	Size selection (300-600bp).]
Size selection (300-600bp).The size selection of the final libraries was done using Agencourt SPRISelect Reagent (Beckman Coulter, Danvers, MA, USA). Selecting for DNA larger than a target size.
?. [16.	Size selection (300-600bp).]
The size selection protocol is based on a starting volume of 100 μl. Adjust the final volume by adding nuclease-free water for a 100 μl total volume.
?. [16.	Size selection (300-600bp).]
Add 60 μl of resuspended AMPure XP beads to the 100 μl. Mix well by pipetting up and down at least 10 times.
?. [16.	Size selection (300-600bp).]
Incubate for  at
20 Room temperature
?. [16.	Size selection (300-600bp).]
Place the tube on an appropriate magnetic stand to separate the beads from the supernatant.After the solution is clear (about 5 minutes), carefully transfer the supernatant containing your DNA to a new tube (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.
?. [16.	Size selection (300-600bp).]
Selecting for DNA smaller than a target size or selecting for DNA within a target region.Add 20 μl resuspended AMPure XP beads to the supernatant, mix well and incubate for  at
20 Room temperature
?. [16.	Size selection (300-600bp).]
Place the tubes on a magnet: allow beads to separate for
?. [16.	Size selection (300-600bp).]
Remove supernatant.
?. [16.	Size selection (300-600bp).]
Add 200 µl80% fresh ethanol to wash the beads, incubate for
?. [16.	Size selection (300-600bp).]
Remove 200 µl 80% EtOH.
?. [16.	Size selection (300-600bp).]
Repeat steps 5.5-5.6.
?. [16.	Size selection (300-600bp).]
Briefly spin the tubes and remove additional ethanol. Ensure any visible quantities of ethanol are removed.
?. [16.	Size selection (300-600bp).]
. Leave the beads in open tubes for
?. [16.	Size selection (300-600bp).]
Add low TE and incubate for .
?. [16.	Size selection (300-600bp).]
Place the tubes on a magnet: allow beads to separate for
?. [16.	Size selection (300-600bp).]
Transfer supernatant into the new tubes.
?. [17.	Agilent 2100 Bioanalyzer.]
Agilent 2100 Bioanalyzer.See point 15.
?. [18. Quantitation of next-generation sequencing (NGS) libraries (NEBNext® Library Quant Kit for Illumina)]
Quantitation      of next-generation sequencing (NGS) libraries (NEBNext® Library Quant Kit      for Illumina)The NEBNext® Library Quant Kit has been optimized to provide substantial performance and workflow improvements to qPCR-based library quantitation.qPCR was performed according to the manufacturer’s instructions.
?. [HiSeq or MiSeq]
Sequencing was performed on Illumina HiSeq 1500 with HiSeq PE Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2 (500 cycles) or Illumina MiSeqwith the MiSeq Reagent Kit V2 (500  cycles) or V3 (600 cycles) according to the manufacturer’s instructions.For HiSeq Rapid Run v2 dilute the pooled sample toin HT1, following HiSeq 1500 loading instructions. For Miseq v3 dilute the pooled sample to  in HT1. For Miseq v2 dilute the pooled sample to  in HT1, following MiSeq loading instructions.