TITLE: A protocol for massively parallel diagnosis and genome sequencing of SARS-CoV-2
AUTHORS: Leigh Monahan, Kay Anantanawat, Joyce To, Aaron Darling

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Managing the current COVID-19 pandemic requires diagnostic testing at an unprecedented scale. However, the crisis has revealed severe deficiencies in our capacity to perform such testing. Here we build on the work of others (1-3) to develop a protocol that not only enables many thousand diagnostic tests to be run in parallel, but also provides near-whole genome sequencing data to facilitate phylogenetic analysis and contact tracing. One of the key features of our protocol is a magnetic bead-based strategy for RNA capture that may circumvent the need for SARS-CoV-2 RNA extraction, currently one of the major bottlenecks in both reagent supply and hands-on sample processing time. This approach also eliminates the requirement for per-sample reverse transcription, significantly reducing per-sample costs. </div><div class = "text-block">The major steps in our wet lab workflow can be summarised as follows. First, we generate a collection of bead-bound, single-stranded DNA probes tiling the entire SARS-CoV-2 genome. Multiple uniquely barcoded probe sets are prepared, each of which are used to capture viral RNA directly from patient swab samples via DNA/RNA hybridisation. Samples are combined together at this stage, enabling cDNA synthesis to be performed in a single pooled reaction. Finally, multiplex PCR is used to generate a library of overlapping amplicons ready for Illumina sequencing. </div><div class = "text-block">A schematic overview of the workflow is attached below, along with a more detailed figure depicting the various stages of library preparation.</div><div class = "text-block"><span style = ":UNDERLINE;">References</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><a href="https://docs.google.com/document/d/1kP2w_uTMSep2UxTCOnUhh1TMCjWvHEY0sUUpkJHPYV4/preview" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://docs.google.com/document/d/1kP2w_uTMSep2UxTCOnUhh1TMCjWvHEY0sUUpkJHPYV4/preview</span></a></li><li style = "counter-reset:ol0;"><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w/abstract" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w/abstract</span></a></li><li style = "counter-reset:ol0;"><a href="https://www.biorxiv.org/content/10.1101/2020.03.20.001008v1.full.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://www.biorxiv.org/content/10.1101/2020.03.20.001008v1.full.pdf</span></a></li></ol></div></div>

[STEPS]
?. [Preparation of barcoded beads for RNA capture]
Wash  of streptavidin-coated Dynabeads™ MyOne™ C1 magnetic beads as per the manufacturer’s instructions:
The first stage in our workflow is the preparation of bead-bound DNA probes that will facilitate subsequent viral RNA capture, cDNA synthesis and multiplex PCR to generate ~200 nt amplicons covering the entire SARS-CoV-2 genome. We use two distinct sets of probes for this, requiring two independent bead preparations for each sample to be processed. Ultimately, this enables library preparation to be performed in two parallel reactions, designed such that neighbouring amplicons do not overlap within the same library PCR. This strategy protects against preferential amplification of short overlap products, and has been successfully developed and employed elsewhere (Quick et al. [2017] Nat Protoc. 12: 1261; https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w/abstract). Throughout the protocol we use the generic terms “pool 1” and “pool 2” to refer to our two capture probe sets, as well as the corresponding primer sets needed for library amplification.  We envisage that capture beads could be prepared in bulk in advance and supplied to testing labs in 96-well or 384-well format. Each uniquely barcoded bead preparation could thus be used for multiple sample batches, provided each batch is sequenced independently. The protocol below is for a small-scale preparation of up to 10 uniquely barcoded bead sets, to be used in development and evaluation of a prototype. We also provide an alternative, lower-cost bead preparation method at the end of this page.
200 µl
?. [Preparation of barcoded beads for RNA capture]
Resuspend the Dynabeads™ magnetic beads in the vial (vortex for >).
?. [Preparation of barcoded beads for RNA capture]
Transfer  of Dynabeads™ magnetic beads to a tube.
200 µl
?. [Preparation of barcoded beads for RNA capture]
Add an equal volume of 1X B&W buffer and resuspend.
?. [Preparation of barcoded beads for RNA capture]
Place the tube on a magnet for  and discard the supernatant.
?. [Preparation of barcoded beads for RNA capture]
Remove the tube from the magnet and resuspend the washed magnetic beads in  of 1X B&W buffer.
200 µl
?. [Preparation of barcoded beads for RNA capture]
Repeat steps 1.4–1.5 twice, for a total of 3 washes.
?. [Preparation of barcoded beads for RNA capture]
Pellet beads once more, and resuspend in  of 2X B&W buffer.
400 µl
?. [Preparation of barcoded beads for RNA capture]
For each unique barcode, take an aliquot of the prepared beads and immobilise an oligonucleotide of the structure 5'-[ biotin ][ spacer ][ partial Illumina p5 adapter ][ sample barcode ][ generic stub ]-3’ onto the beads as follows:
?. [Preparation of barcoded beads for RNA capture]
Add  of 5 μM oligonucleotide ([Biotin]-barcoded bead tether) to  of washed beads.
40 µl
40 µl
?. [Preparation of barcoded beads for RNA capture]
Incubate for  at room temperature using gentle rotation.
?. [Preparation of barcoded beads for RNA capture]
Separate the biotinylated DNA coated beads with a magnet for 2–3 min.
?. [Preparation of barcoded beads for RNA capture]
Wash the coated beads 2–3 times with  1X B&W buffer.
100 µl
?. [Preparation of barcoded beads for RNA capture]
Resuspend in  of nuclease-free water.
120 µl
?. [Preparation of barcoded beads for RNA capture]
For each barcoded sample, generate two distinct libraries of bead-bound SARS-CoV-2 probes by annealing separate oligonucleotide pools with the general structure 3'-[ generic stub complement ][ SARS-CoV-2 target ]-5’ and performing strand extension:
?. [Preparation of barcoded beads for RNA capture]
Mix the following components in 0.2 mL PCR tubes:        Component                                                                     Pool 1                   Pool 2        Bead-bound template (previous step)                              5X PrimeSTAR GXL buffer                                                  dNTP mixture (2.5 mM each)                                             SARS-CoV-2 probe pool 1 or 2 (10 μM)                            PrimeSTAR GXL DNA polymerase
60 µl
60 µl
20 µl
20 µl
8 µl
8 µl
10 µl
10 µl
2 µl
2 µl
?. [Preparation of barcoded beads for RNA capture]
Incubate the reaction as follows:        Step                             Temperature                Time                                Cycles        Denaturation                                1        Annealing                                     1        Extension                                      1        Hold                                                       Indefinite                         1
98 °C
55 °C
68 °C
4 °C
?. [Preparation of barcoded beads for RNA capture]
For each bead sample, dissociate and remove the non-biotinylated strand as follows:
?. [Preparation of barcoded beads for RNA capture]
Pellet the beads on a magnet and wash once in  1X SSC.
50 µl
?. [Preparation of barcoded beads for RNA capture]
Resuspend the beads in  of freshly prepared 0.15 M NaOH and incubate at room temperature for .
20 µl
?. [Preparation of barcoded beads for RNA capture]
Pellet the beads on a magnet and discard the supernatant containing the non-biotinylated strand.
?. [Preparation of barcoded beads for RNA capture]
Wash the beads once with  0.1M NaOH, once with  of 1X B&W buffer and once with  TE buffer.
50 µl
50 µl
50 µl
?. [Preparation of barcoded beads for RNA capture]
Resuspend beads in  TE buffer.
50 µl
The above steps are designed to produce two libraries of single-stranded, bead-bound SARS-CoV-2 probes for each sample barcode, with the general structure 5'-[ biotin ][ spacer ][ partial Illumina p5 adapter ][ sample barcode ][ generic stub ][ SARS-CoV-2 target ]-3’. Once the method has been suitably tested and is ready for deployment at scale, we envisage that these steps could be simplified substantially by synthesising the biotinylated probe sets in full for each sample barcode and immobilising these directly onto the streptavidin-coated beads.
?. [Sample collection and preparation]
Collect nasopharyngeal swabs in a  volume of transport medium.
1.5 ml
We are currently testing a range of media with the aim of identifying a formulation that optimally preserves viral RNA and supports hybridisation with our bead-bound probes in the following step. These include UTM, Lysis/binding buffer and a combination of the two (see Materials). Alternatively, kit-extracted RNA or synthetic control RNA can be used.
?. [Sample collection and preparation]
Quantify  of swab diluent using the Qubit RNA HS assay or similar.
10 µl
?. [RNA capture]
Alternatively, use multi-well PCR plates. For 384-well plates, sample volumes will need to be scaled down in subsequent steps.
25 µl
Alternatively, use multi-well PCR plates. For 384-well plates, sample volumes will need to be scaled down in subsequent steps.
?. [RNA capture]
Pellet the beads on a magnet and discard the supernatant.
?. [RNA capture]
Add  of swab diluent directly to each bead pellet (i.e. 2 x  per swab sample)  and resuspend completely by pipette mixing. Alternatively, use purified or synthetic control RNA in a buffer suitable for probe hybridisation e.g. 1X SSC.
50 µl
50 µl
?. [RNA capture]
Place reactions in a preheated thermal cycler with a heated lid and incubate at  for , then ramp to  at a rate of -0.1℃/s and proceed immediately to the next step.
75 °C
25 °C
?. [RNA capture]
Pellet the beads on a magnet and discard the supernatant.
?. [RNA capture]
Resuspend each reaction in  of Washing buffer A.
20 µl
?. [RNA capture]
Combine all “pool 1” samples together into a fresh tube and repeat this process for “pool 2”.
For large sample batches it should only be necessary to pool a small aliquot of each reaction. The combined samples will be used as input for cDNA synthesis. We are currently testing whether there is a limit to the amount of beads that can be tolerated in the reverse transcription reaction, as well as the subsequent library amplification.
?. [cDNA synthesis]
Wash each pooled bead sample once with an equivalent volume of Washing buffer A and twice with the same volume of Washing buffer B, using a magnet to separate the beads from the solution between each washing step. After the final wash, leave the beads suspended in Washing buffer B at room temperature and proceed immediately to the next step.
?. [cDNA synthesis]
Prepare a reverse transcriptase master mix by combining the following components from the SuperScript™ IV First-Strand Synthesis System:        Component                                                        Volume        5X SSIV buffer                                                          10 mM dNTP mix (10 mM each)                           100 mM DTT                                                             Ribonuclease inhibitor (40 U/μL)                           DEPC-treated water
20 µl
5 µl
5 µl
5 µl
60 µl
?. [cDNA synthesis]
Pellet beads from step 14 on a magnet and discard the supernatant. Pulse spin tubes for 5 s, return to the magnet and discard any remaining liquid to ensure all the Washing buffer B has been removed (residual buffer may inhibit the reverse transcriptase reaction).
?. [cDNA synthesis]
Perform one final wash in  of reverse transcriptase master mix. Again pellet beads on a magnet and discard the supernatant.
25 µl
?. [cDNA synthesis]
Resuspend each pooled bead sample in  of reverse transcriptase master mix and add  of SuperScript™ IV Reverse Transcriptase (200 U/μL). Mix well by pipetting and briefly centrifuge.
19 µl
1 µl
?. [cDNA synthesis]
Incubate the reactions at  for .
55 °C
?. [cDNA synthesis]
Inactivate by incubating at  for .
80 °C
?. [cDNA synthesis]
Pellet beads by placing each tube on a magnet. Discard the supernatant and wash once in TE buffer.
?. [Library amplification]
Pellet the “pool 1” and “pool 2” cDNA-coated bead samples from the previous step on a magnet, discard the supernatant and resuspend each in  of nuclease-free water.
Here we generate amplicon libraries from each pooled cDNA sample by multiplex PCR. We also incorporate Illumina sequencing adapters and additional barcodes at this stage, producing sequence-ready samples that could potentially be combined with additional library pools for further scalability. Sequencing adapters are added over two sequential rounds of PCR, although we envisage that a single step protocol may also be feasible.
28 µl
?. [Library amplification]
Combine the first-round PCR components in separate 0.2 mL PCR tubes:        Component                                                                Pool 1                       Pool 2        cDNA-coated beads (pool 1 or 2)                                                5X Q5 reaction buffer                                                                        10 mM dNTPs                                                                                      Illumina P5 index primer (10 μM)                                                  SARS-CoV-2 primer pool 1 or 2 (10 μM)                                       Q5 Hot Start High-Fidelity Polymerase
26.7 µl
26.7 µl
10 µl
10 µl
1 µl
1 µl
2.5 µl
2.5 µl
9.3 µl
9.3 µl
0.5 µl
0.5 µl
The SARS-CoV-2 primer pools contain 124 (pool 1) and 123 (pool 2) individual primer sequences. The concentration of each primer in the final reaction is 0.015 μM, as recommended in previous studies (Quick et al. [2017] Nat Protoc. 12: 1261).
?. [Library amplification]
Mix well by pipetting and briefly centrifuge.
?. [Library amplification]
Incubate the first-round PCR reactions as follows:        Step                                                Temperature                 Time                            Cycles        Heat activation                                            1        Denaturation                                                 25-35        Annealing/extension                                  25-35        Hold                                                                          Indefinite                      1
98 °C
98 °C
65 °C
4 °C
?. [Library amplification]
For each reaction, pellet the cDNA-coated beads on a magnet and transfer the supernatant containing the amplified product to a new tube.
?. [Library amplification]
Clean up each reaction by performing a left side size selection with a 1.2X volume of SPRIselect beads as per the manufacturer’s instructions (Beckman Coulter). Elute the sample in  of nuclease-free water.
40 µl
?. [Library amplification]
Quantify  of each purified sample using the Qubit dsDNA HS assay or similar.
1 µl
?. [Library amplification]
Combine the second-round PCR components in separate 0.2 mL PCR tubes:        Component                                                                Pool 1                        Pool 2        Purified template (previous step)                                                    5X PrimeSTAR GXL buffer                                                                 dNTP mixture (2.5 mM each)                                                              Illumina P5 flow cell primer (10 μM)                                            Illumina P7 index primer (10 μM)                                                 PrimeSTAR GXL DNA polymerase                                                      Nuclease-free water                                                 to                 to
10 ng
10 ng
10 µl
10 µl
4 µl
4 µl
1.25 µl
1.25 µl
1.25 µl
1.25 µl
1 µl
1 µl
50 µl
50 µl
?. [Library amplification]
Mix well by pipetting and briefly centrifuge.
?. [Library amplification]
Incubate the second-round PCR reactions as follows:        Step                                    Temperature                 Time                             Cycles        Denaturation                                                            6            Annealing                                                                 6        Extension                                                                 6        Hold                                                             Indefinite                      1
98 °C
60 °C
68 °C
4 °C
?. [Library amplification]
Clean up each reaction by performing a left side size selection with a 0.8X volume of SPRIselect beads as per the manufacturer’s instructions (Beckman Coulter). Elute the sample in  of nuclease-free water.
40 µl
?. [Library quantitation and sequencing]
Determine the concentration of the final “pool 1” and “pool 2” multiplexed libraries in ng/μL using the Qubit dsDNA HS assay or similar.
?. [Library quantitation and sequencing]
Assess the fragment size distribution of each library and determine the average fragment length using an Agilent 2100 Bioanalyzer or similar.
?. [Library quantitation and sequencing]
Calculate sample molarity using the following formula:        Concentration in nM = [(concentration in ng/μL) / (660 g/mol x average size in bp)] x 106
?. [Library quantitation and sequencing]
Combine an equimolar amount of each library and mix.
If sequencing additional samples, quantitate them as above and combine at the desired relative molarity.
?. [Library quantitation and sequencing]
Sequence the final pooled library using an Illumina instrument, generating 2 x 150 nt paired end reads
?. [Alternative bead preparation method]
Wash  of streptavidin-coated Dynabeads™ MyOne™ C1 magnetic beads as per the manufacturer’s instructions:
This alternative method enables the production of multiple uniquely barcoded bead preparations from a single biotinylated template oligo. This reduces oligo synthesis costs, but does introduce an additional level of protocol complexity.
200 µl
?. [Alternative bead preparation method]
Resuspend the Dynabeads™ magnetic beads in the vial (vortex for >).
?. [Alternative bead preparation method]
Transfer  of Dynabeads™ magnetic beads to a tube.
200 µl
?. [Alternative bead preparation method]
Add an equal volume of 1X B&W buffer and resuspend.
?. [Alternative bead preparation method]
Place the tube on a magnet for  and discard the supernatant.
?. [Alternative bead preparation method]
Remove the tube from the magnet and resuspend the washed magnetic beads in  of 1X B&W buffer.
200 µl
?. [Alternative bead preparation method]
Repeat steps 38.4–38.5 twice, for a total of 3 washes.
?. [Alternative bead preparation method]
Pellet beads once more, and resuspend in  of 2X B&W buffer.
400 µl
?. [Alternative bead preparation method]
Immobilise a generic oligonucleotide of the structure 5'-[ biotin ][ spacer ][ partial Illumina p5 adapter ]-3’ onto the beads as follows:
?. [Alternative bead preparation method]
Add  of 5 μM oligonucleotide ([Biotin]-bead tether) to  of washed beads.
400 µl
400 µl
?. [Alternative bead preparation method]
Incubate for  at room temperature using gentle rotation.
?. [Alternative bead preparation method]
Separate the biotinylated DNA coated beads with a magnet for 2–3 min.
?. [Alternative bead preparation method]
Wash the coated beads 2–3 times with 1X B&W buffer.
?. [Alternative bead preparation method]
Resuspend in  of nuclease-free water.
1.2 ml
?. [Alternative bead preparation method]
For each unique barcode, generate two libraries of bead-bound SARS-CoV-2 probes via a two-stage annealing and extension process using a barcoded linker oligonucleotide of the structure 3'-[ partial Illumina p5 adapter  complement ][ sample barcode ][ generic stub complement ]-5’ and two separate oligonucleotide pools with the general structure 3'-[ generic stub complement ][ SARS-CoV-2 target ]-5’:
?. [Alternative bead preparation method]
Mix the following components in a 0.2 mL PCR tube:        Component                                                                     Pool 1                   Pool 2        Bead-bound template (previous step)                             5X PrimeSTAR GXL buffer                                                 dNTP mixture (2.5 mM each)                                            Barcoded linker (10 μM)                                                     SARS-CoV-2 probe pool 1 or 2 (10 μM)                            PrimeSTAR GXL DNA polymerase
60 µl
60 µl
20 µl
20 µl
8 µl
8 µl
5 µl
5 µl
5 µl
5 µl
2 µl
2 µl
?. [Alternative bead preparation method]
Incubate the reaction as follows:        Step                             Temperature                 Time                                Cycles        Denaturation                                4        Annealing                                     4        Extension                                      4        Hold                                                       Indefinite                         4
98 °C
55 °C
68 °C
4 °C
?. [Alternative bead preparation method]
For each bead sample, dissociate and remove the non-biotinylated strand as follows:
?. [Alternative bead preparation method]
Pellet the beads on a magnet and wash once in  1X SSC.
50 µl
?. [Alternative bead preparation method]
Resuspend the beads in  of freshly prepared 0.15 M NaOH and incubate at room temperature for .
20 µl
?. [Alternative bead preparation method]
Pellet the beads on a magnet and discard the supernatant containing the non-biotinylated strand.
?. [Alternative bead preparation method]
Wash the beads once with  0.1M NaOH, once with  of 1X B&W buffer and once with  TE buffer.
50 µl
50 µl
50 µl
?. [Alternative bead preparation method]
Resuspend beads in  TE buffer.
50 µl