TITLE: SARS-CoV-2 Genome Sequencing Using Long Pooled Amplicons on Illumina Platforms
AUTHORS: John-Sebastian Eden, Eby Sim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes methods to sequence SARS-CoV-2 with pooled amplicons (14 x 2.5kb) using Illumina Platforms.</div></div>

[STEPS]
?. [Viral RNA extraction]
Add  to each 200 μl sample and mix well by gentle vortexing.
[Viral RNA Buffer]
?. [Viral RNA extraction]
Transfer the mixture into a Zymo-Spin IC column placed in a collection tube. Centrifuge at  for  to bind viral RNA to matrix.
Centrifuge: 12000 34
?. [Viral RNA extraction]
Transfer the column into a new collection tube.
?. [Viral RNA extraction]
Add  to the column, centrifuge at  for .
[Viral Wash Buffer]
Centrifuge: 12.000 34
?. [Viral RNA extraction]
Transfer the column into a new collection tube.
?. [Viral RNA extraction]
Repeat steps 6 & 7:
?. [Viral RNA extraction]
Add  to the column and centrifuge at  for  to ensure complete removal of the wash buffer.
[Ethanol (95-100%)]
Centrifuge: 12.000 34
?. [Viral RNA extraction]
Transfer the column into a clean elution tube (2 ml Eppendorf PCR clean, DNA LoBind tube).
?. [Viral RNA extraction]
Add  directly to the column matrix, incubate for  and then centrifuge at  for  to elute the viral RNA.
[DNase/RNase-Free Water]
Centrifuge: 12.000 34
?. [Viral RNA extraction]
The eluted viral RNA can be used immediately or stored frozen at .
-80 °C
?. [Viral RNA extraction]
Combine  with 100 μl respiratory sample (sputum, aspirate or swab medium), then mix well by gentle vortexing.
[2X DNA/RNA Shield]
?. [Viral RNA extraction]
Add  to the column, centrifuge at  for .
[Viral Wash Buffer]
Centrifuge: 12.000 34
?. [Viral RNA extraction]
Transfer the column into a new collection tube.
?. [cDNA synthesis]
cDNA is prepared from viral RNA using SuperScript IV VILO Master Mix, which combines both random and oligo-dT priming for first strand synthesis. The oligo-dT priming is essential as the reverse primer for the final amplicon sits close to the viral poly-A tail in the 3’ UTR. When using individual components to set up cDNA synthesis instead of the VILO mastermix, use the recommended protocol for a random hexamer primed reaction but supplement the reaction with oligo-dT so the final random hexamer/oligo-dT ratio is 3:1.
?. [Viral RNA extraction]
Here we provide an example low throughput viral RNA extraction protocol; however, any platform that extracts viral or pathogen RNA (or total nucleic acid) should be suitable. We typically use the residual viral extracts from clinical samples following diagnostic RT-PCRs, which include extracts off both Qiagen BioRobot EZ1 and Roche MagnaPure 96 platforms. Please ensure appropriate PPE and containment to avoid exposure to infectious samples, noting that following the addition of the DNA/RNA Shield, the virus will be inactivated.
?. [cDNA synthesis]
Setup the following reaction mix for each sample and a no template control (NTC):  AB1REAGENTVOLUME (μl)2SuperScript IV VILO MasterMix (5X)53Nuclease free water154Viral RNA55TOTAL25
AB1REAGENTVOLUME (μl)2SuperScript IV VILO MasterMix (5X)53Nuclease free water154Viral RNA55TOTAL25
?. [cDNA synthesis]
Incubate the reaction as follows:  ABC1STEPTEMP (°C)TIME (mm:ss)2Priming2510:003Extension5020:004Denature RT8505:005Hold4∞
ABC1STEPTEMP (°C)TIME (mm:ss)2Priming2510:003Extension5020:004Denature RT8505:005Hold4∞
?. [cDNA synthesis]
Keep the cDNA  if being used immediately or store frozen at .
on ice
-20 °C
?. [Genome Amplification]
Next we take the cDNA prepared from viral RNA and amplify 14 regions (~2.5kb each) that tile across the viral genome. The PCRs are performed in parallel and is NOT a multiplexed reaction. The general approach is demonstrated by the following schematic:
?. [Genome Amplification]
The amplicon primers are as follows:  ABCD1PCRPRIMERSEQUENCE (5' - 3')LENGTH (nt)2A1SARS2_A1F_31ACCAACCAACTTTCGATCTCTTGT25623SARS2_A1R_2569GCTTCAACAGCTTCACTAGTAGGT4A2SARS2_A2F_4295ACAGTGCTTAAAAAGTGTAAAAGTGCC25795SARS2_A2R_6847ACAGTATTCTTTGCTATAGTAGTCGGC6A3SARS2_A3F_8596ACTTGTGTTCCTTTTTGTTGCTGC24797SARS2_A3R_11049GAACAAAGACCATTGAGTACTCTGGA8A4SARS2_A4F_12711TACGACAGATGTCTTGTGCTGC25369SARS2_A4R_15225TAACATGTTGTGCCAACCACCA10A5SARS2_A5F_16847ACTATGGTGATGCTGTTGTTTACCG243211SARS2_A5R_19254ACCAGGCAAGTTAAGGTTAGATAGC12A6SARS2_A6F_21358ACAAATCCAATTCAGTTGTCTTCCTATTC249013SARS2_A6R_23823TGTGTACAAAAACTGCCATATTGCA14A7SARS2_A7F_25602ACTAGCACTCTCCAAGGGTGTT257115SARS2_A7R_28146AGGTTCCTGGCAATTAATTGTAAAAGG16B1SARS2_B1F_1876ATCAGAGGCTGCTCGTGTTGTA257517SARS2_B1R_4429AGTTTCCACACAGACAGGCATT18B2SARS2_B2F_6287TGGTGTATACGTTGTCTTTGGAGC256519SARS2_B2R_8828CACTTCTCTTGTTATGACTGCAGC20B3SARS2_B3F_10363TGTTCGCATTCAACCAGGACAG244021SARS2_B3R_12780CCTACCTCCCTTTGTTGTGTTGT22B4SARS2_B4F_14546AGGAATTACTTGTGTATGCTGCTGA260723SARS2_B4R_17131ACACTATGCGAGCAGAAGGGTA24B5SARS2_B5F_18897TGTTAAGCGTGTTGACTGGACT255925SARS2_B5R_21428TGACCTTCTTTTAAAGACATAACAGCAG26B6SARS2_B6F_23123CCAGCAACTGTTTGTGGACCTA255127SARS2_B6R_25647AGGTGTGAGTAAACTGTTACAAACAAC28B7SARS2_B7F_27447TCACTACCAAGAGTGTGTTAGAGGT242029SARS2_B7R_29837TTCTCCTAAGAAGCTATTAAAATCACATGG
ABCD1PCRPRIMERSEQUENCE (5' - 3')LENGTH (nt)2A1SARS2_A1F_31ACCAACCAACTTTCGATCTCTTGT25623SARS2_A1R_2569GCTTCAACAGCTTCACTAGTAGGT4A2SARS2_A2F_4295ACAGTGCTTAAAAAGTGTAAAAGTGCC25795SARS2_A2R_6847ACAGTATTCTTTGCTATAGTAGTCGGC6A3SARS2_A3F_8596ACTTGTGTTCCTTTTTGTTGCTGC24797SARS2_A3R_11049GAACAAAGACCATTGAGTACTCTGGA8A4SARS2_A4F_12711TACGACAGATGTCTTGTGCTGC25369SARS2_A4R_15225TAACATGTTGTGCCAACCACCA10A5SARS2_A5F_16847ACTATGGTGATGCTGTTGTTTACCG243211SARS2_A5R_19254ACCAGGCAAGTTAAGGTTAGATAGC12A6SARS2_A6F_21358ACAAATCCAATTCAGTTGTCTTCCTATTC249013SARS2_A6R_23823TGTGTACAAAAACTGCCATATTGCA14A7SARS2_A7F_25602ACTAGCACTCTCCAAGGGTGTT257115SARS2_A7R_28146AGGTTCCTGGCAATTAATTGTAAAAGG16B1SARS2_B1F_1876ATCAGAGGCTGCTCGTGTTGTA257517SARS2_B1R_4429AGTTTCCACACAGACAGGCATT18B2SARS2_B2F_6287TGGTGTATACGTTGTCTTTGGAGC256519SARS2_B2R_8828CACTTCTCTTGTTATGACTGCAGC20B3SARS2_B3F_10363TGTTCGCATTCAACCAGGACAG244021SARS2_B3R_12780CCTACCTCCCTTTGTTGTGTTGT22B4SARS2_B4F_14546AGGAATTACTTGTGTATGCTGCTGA260723SARS2_B4R_17131ACACTATGCGAGCAGAAGGGTA24B5SARS2_B5F_18897TGTTAAGCGTGTTGACTGGACT255925SARS2_B5R_21428TGACCTTCTTTTAAAGACATAACAGCAG26B6SARS2_B6F_23123CCAGCAACTGTTTGTGGACCTA255127SARS2_B6R_25647AGGTGTGAGTAAACTGTTACAAACAAC28B7SARS2_B7F_27447TCACTACCAAGAGTGTGTTAGAGGT242029SARS2_B7R_29837TTCTCCTAAGAAGCTATTAAAATCACATGG
?. [Genome Amplification]
Prepare the primers for each amplicon set by combining ,  and  into clean PCR strips, as below:
[forward primer (100 μM)]
[reverse primer (100 μM)]
[nuclease free water]
?. [Genome Amplification]
One standard RT-PCR is shown below, but each sample requires 14 reactions in total, each containing the different amplicon primers A1-A7 & B1-B7.  AB1REAGENTVOLUME (μl)2Platinum SuperFi Green Mastermix (2X)103Nuclease free water6.54Primer pool (5 μM)25Viral cDNA1.56TOTAL20
AB1REAGENTVOLUME (μl)2Platinum SuperFi Green Mastermix (2X)103Nuclease free water6.54Primer pool (5 μM)25Viral cDNA1.56TOTAL20
?. [Genome Amplification]
Setup the following reaction mix for each sample and the no template control (NTC) according to requirements (one sample has 14.5 reactions, 14 primers + 0.5 for pipetting error):  ABCDE1REAGENTVOLUME (μl), s=samples2S=1S=3S=6S=123Platinum SuperFi Green Mastermix (2X)14543587017404Nuclease free water94.25282.75565.51131
ABCDE1REAGENTVOLUME (μl), s=samples2S=1S=3S=6S=123Platinum SuperFi Green Mastermix (2X)14543587017404Nuclease free water94.25282.75565.51131
?. [Genome Amplification]
Dispense  into each well as required, a suggested plate setup is provided below for a 6 sample plate:
[reaction mix]
?. [Genome Amplification]
Using a 8-channel multipipettor, add  into each well according to the plate layout.
[viral cDNA]
?. [Genome Amplification]
Seal the plate, and centrifuge for  in a plate spinner.
?. [Genome Amplification]
Incubate the reaction as follows:  ABCD1STEPTEMP (°C)TIME (mm:ss)CYCLES2Hot start9802:001X3Denaturation9800:1540X4Annealing6500:305Extension7202:006Final extension7205:001X7Hold4∞
ABCD1STEPTEMP (°C)TIME (mm:ss)CYCLES2Hot start9802:001X3Denaturation9800:1540X4Annealing6500:305Extension7202:006Final extension7205:001X7Hold4∞
?. [Genome Amplification]
Analyse  on a 1% agarose gel with DNA staining dye. When using Invitrogen E-Gel 48 Agarose precast gels, preload each well with 10 μl of EB buffer. All bands are expected to be ~2.5kb, so run with an appropriate DNA ladder. To aid in pooling visualisation, it is preferable to run all set A and B reactions for one sample on the same row.
[each RT-PCR reaction]
?. [PCR pooling and cleanup]
An example gel is provided below for two different samples:
?. [PCR pooling and cleanup]
Pool the individual amplicons for each sample into a single well of a clean plate or PCR strip. The aim here is to roughly balance the amount of DNA from each amplicon to provide even coverage across the genome. Target the final volume in the pool to be ~  (i.e.  for all 14 amplicons when yield is even).
40 µl
3 µl
?. [PCR pooling and cleanup]
Adjust the final volume of the pooled PCR product to  by aliquoting when exceeding or adding an appropriate volume of clean EB buffer when less.
50 µl
?. [PCR pooling and cleanup]
Add  (0.8X bead ratio). Gently pipette the entire volume up and down 15 times to mix thoroughly.
[room temperature AMPure XP beads]
?. [PCR pooling and cleanup]
Incubate at  for .
0 Room temperature
?. [PCR pooling and cleanup]
Place the plate/tubes onto the magnetic stand for at least , until the solution appears clear. Do not remove from magnetic stand during washing steps, and take care to not disturb beads.
?. [PCR pooling and cleanup]
Remove and discard the supernatant from each well by pipetting.
?. [PCR pooling and cleanup]
Add  to each well without disturbing the beads.
[freshly prepared 80% EtOH]
?. [PCR pooling and cleanup]
Incubate at  for .
0 Room temperature
?. [PCR pooling and cleanup]
Remove and discard the supernatant from each well by pipetting.
?. [PCR pooling and cleanup]
Repeat steps 35 to 37 for a total of two EtOH washes:
?. [PCR pooling and cleanup]
Allow the beads to air dry for  to . Visually inspect wells to ensure any small droplets are completely removed by pipetting or evaporation.
?. [PCR pooling and cleanup]
Remove the plate/tubes from the magnetic stand.
?. [PCR pooling and cleanup]
Resuspend the dried bead pellet with .
[EB buffer]
?. [PCR pooling and cleanup]
Gently pipette the entire volume up and down 15 times to mix thoroughly.
?. [PCR pooling and cleanup]
Place the plate/tubes onto the magnetic stand at room temperature for at least , until the solution appears clear.
?. [PCR pooling and cleanup]
Transfer the cleared supernatant containing the purified DNA into a suitable plate, strip or tube. Use immediately or store frozen at .
-20 °C
?. [PCR pooling and cleanup]
Quantify all purified DNA using the Qubit dsDNA broad range assay.
?. [PCR pooling and cleanup]
For any samples >10ng/μl, roughly dilute using EB buffer so the final concentration is between  to .
?. [PCR pooling and cleanup]
Re-quantify all purified and adjusted DNA using the Qubit dsDNA high sensitivity assay.
?. [PCR pooling and cleanup]
Dilute to  using EB buffer (ensure at least ) and proceed to library prep.
[total volume]
?. [PCR pooling and cleanup]
Add  to each well without disturbing the beads.
[freshly prepared 80% EtOH]
?. [PCR pooling and cleanup]
Incubate at  for .
0 Room temperature
?. [PCR pooling and cleanup]
Remove and discard the supernatant from each well by pipetting.
?. [DNA library preparation and sequencing]
The following protocol follows the Illumina Nextera XT DNA library prep kit except for two important changes: i) all reaction volumes are halved to save on reagents and ii) that following the clean up after library amplification, the libraries are manually normalised rather than with the provided normalisation beads.
?. [DNA library preparation and sequencing]
Combine  and  for each sample into a PCR plate or strip.
[Tagment DNA Buffer (TD)]
[pooled, diluted (0.2 ng/μl) amplicons]
?. [DNA library preparation and sequencing]
Mix well by pipetting 10 times.
?. [DNA library preparation and sequencing]
Add  to each well on top of the TD/DNA mix.
[Amplicon Tagment Mix (ATM)]
?. [DNA library preparation and sequencing]
Mix well by pipetting 10 times. Seal and briefly centrifuge.
?. [DNA library preparation and sequencing]
Incubate the reaction mix as follows:  ABC1STEPTEMP (°C)TIME (mm:ss)2Tagmentation5505:003Hold10∞
ABC1STEPTEMP (°C)TIME (mm:ss)2Tagmentation5505:003Hold10∞
?. [DNA library preparation and sequencing]
Following tagmentation, immediately remove the reaction from the thermocycler and add  to stop the reaction.
[Neutralize Tagment Buffer (NT)]
?. [DNA library preparation and sequencing]
Mix well by pipetting 10 times. Seal and briefly centrifuge.
?. [DNA library preparation and sequencing]
Incubate at  for .
-20 Room temperature
?. [DNA library preparation and sequencing]
Add indices and Nextera PCR Master Mix (NPM) to the neutralised tagmentation reaction for each sample as below:  AB1REAGENTVOLUME (μl)2Neutralised tagmentation reaction12.53i7 adapter2.54i5 adapter2.55Nextera PCR Master Mix7.56TOTAL25
AB1REAGENTVOLUME (μl)2Neutralised tagmentation reaction12.53i7 adapter2.54i5 adapter2.55Nextera PCR Master Mix7.56TOTAL25
?. [DNA library preparation and sequencing]
Incubate the reaction as follows:  ABCD1STEPTEMP (°C)TIME (mm:ss)CYCLES2Hot start7203:001X3Initial denature9500:301X4Denaturation9500:1012X5Annealing5500:306Extension7200:307Final extension7205:001X8Hold10∞
ABCD1STEPTEMP (°C)TIME (mm:ss)CYCLES2Hot start7203:001X3Initial denature9500:301X4Denaturation9500:1012X5Annealing5500:306Extension7200:307Final extension7205:001X8Hold10∞
?. [DNA library preparation and sequencing]
Add  (0.6X bead ratio) to the 25 μl of amplified libraries. Gently pipette the entire volume up and down 15 times to mix thoroughly.
[room temperature AMPure XP beads]
?. [DNA library preparation and sequencing]
Incubate at  for .
-20 Room temperature
?. [DNA library preparation and sequencing]
Place the plate/tubes onto the magnetic stand for at least , until the solution appears clear. Do not remove from magnetic stand during washing steps, and take care to not disturb beads.
?. [DNA library preparation and sequencing]
Remove and discard the supernatant from each well by pipetting.
?. [DNA library preparation and sequencing]
Add  to each well without disturbing the beads.
[freshly prepared 80% EtOH]
?. [DNA library preparation and sequencing]
Incubate at  for .
-20 Room temperature
?. [DNA library preparation and sequencing]
Remove and discard the supernatant from each well by pipetting.
?. [DNA library preparation and sequencing]
Repeat steps 64 to 66 for a total of two EtOH washes:
?. [DNA library preparation and sequencing]
Allow the beads to air dry for  to . Visually inspect wells to ensure any small droplets are completely removed by pipetting or evaporation.
?. [DNA library preparation and sequencing]
Remove the plate/tubes from the magnetic stand.
?. [DNA library preparation and sequencing]
Resuspend the dried bead pellet with .
[EB buffer]
?. [DNA library preparation and sequencing]
Gently pipette the entire volume up and down 15 times to mix thoroughly.
?. [DNA library preparation and sequencing]
Place the plate/tubes onto the magnetic stand at room temperature for at least , until the solution appears clear.
?. [DNA library preparation and sequencing]
Transfer  (containing the purified DNA) into a suitable plate, strip or tube. Use immediately or store frozen at .
[cleared supernatant]
-20 °C
?. [DNA library preparation and sequencing]
Quantify all purified DNA using the Qubit dsDNA high sensitivity assay.
?. [DNA library preparation and sequencing]
Pool the individual libraries equally in DNA amount based on the Qubit values. Here, we assume the libraries will have similar fragment lengths and distributions.
?. [DNA library preparation and sequencing]
Quantify the final pool of libraries using the Qubit dsDNA high sensitivity assay.
?. [DNA library preparation and sequencing]
Analyse  with Agilent High Sensitivity D5000 Screen Tape ensuring the whole fragment peak is captured.
[final pool of libraries]
?. [DNA library preparation and sequencing]
Scale the calculated molarity from the Tapestation to the Qubit DNA concentration using the following formula:             Final molarity = (Tapestation DNA molarity) x (Qubit DNA conc / Tapestation DNA conc)
?. [DNA library preparation and sequencing]
Dilute the final pooled libraries down to   (at least 50μl) using EB buffer, and add PhiX sequencing control if required (we normally don't).
?. [DNA library preparation and sequencing]
Combine  and  to dilute the final pool of libraries to  for loading.
[of 1nM library pool]
[of EB buffer]
?. [DNA library preparation and sequencing]
Load  into a defrosted Illumina iSeq cartridge with flow cell, and sequence with at least 75 nt paired end sequencing.
[of 0.2nM library pool]
Assuming even pooling, each run can comfortably include 12-18 SARS-CoV-2 genome libraries with >1000x coverage for consensus calling.
?. [DNA library preparation and sequencing]
Finally, assemble your viruses and upload them onto www.gisaid.org. Some more details on basic bioinformatic workflow will be provided soon.
?. [DNA library preparation and sequencing]
Add  to each well without disturbing the beads.
[freshly prepared 80% EtOH]
?. [DNA library preparation and sequencing]
Incubate at  for .
-20 Room temperature
?. [DNA library preparation and sequencing]
Remove and discard the supernatant from each well by pipetting.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Due to the larger amplicon size generated from this protocol, the application of Nanopore sequencing using the Rapid Barcoding kit was tested. The input for this workflow are four individual pooled samples that have been cleaned and quantified. The output of this process is a “pseudomolecule” with the variant bases of sequenced sample relative to the genome of MN908947.3
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Perform Flow cell QC as per Oxford Nanopore’s instructions prior to starting library preparation.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
For each sample, combine between 100-200 fmol of pooled amplicons with .
[fragmentation mix]
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Adjust the final volume for each tube to  using Nuclease free water. Gently flick to mix and spin down.
10 µl
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Incubate tubes at  for  and then at  for  in a thermocycler. Place tubes  to cool.
30 °C
80 °C
on ice
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Pool all samples into a 1.5 ml LoBind tube. Gently flick to mix and spin down.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Aspirate and dispense  into a different 1.5 ml LoBind tube and add . Gently flick to mix and spin down.
[of pooled RBK004 libraries]
[RAP]
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Incubate the tube at  for .
0 Room temperature
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Store the prepared library  until required.
on ice
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Perform priming and loading the Spot ON Flow Cell as per Oxford Nanopore’s instructions.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Perform sequencing for up to  or until enough data is obtained.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Once sequencing run is completed, perform base calling, demultiplexing and adaptor trimming using Guppy.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Filter each demultiplexed read set using NanoFilt (https://github.com/wdecoster/nanofilt) to keep reads with a minimum quality of 10 and a maximum length of 2,700 bases.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Map each filtered read set to onto the reference genome (accession: MN908947.3) separately using Minimap2 (https://github.com/lh3/minimap2) and convert the resulting mapping file into a sorted .bam file using SAMtools (https://github.com/samtools/samtools).
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Visualise and inspect the mapping profile over the reference genome.
N.B.  Expect no/low coverage (
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Perform variant calling using medaka_variant (https://github.com/nanoporetech/medaka).
Ensure that you select the correct medaka model based on your Nanopore sequencing platform. In addition, variants with a QUAL score of less than 30 should be interrogated before generating a consensus pseudomolecule.
?. [Addendum: Sequencing pooled amplicons with Oxford Nanopore Rapid Barcoding kit (SQK-RBK004)]
Generate consensus sequence from the resulting .vcf file using BCFtools (https://samtools.github.io/bcftools/).
?. [Genome Amplification]
Using a 8-channel multipipettor, add  into each well according to the plate layout.
[of the diluted and premixed primer sets]