TITLE: SARS-CoV-2 detection with ApharSeq
AUTHORS: Daphna Strauss, Ayelet Rahat, Israa Sharkia, Alon Chappleboim, Miriam Adam, Daniel Kitsberg, Gavriel Fialkoff, Matan Lotem, Omer Gershon, Anna-Kristina Schmidtner, Esther Oiknine-Djian, Agnes Klochendler, Ronen Sadeh, Yuval Dor, Dana Wolf, Naomi Habib, Nir Friedman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">The global SARS-CoV-2 pandemic led to a steep increase in the need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays that involve a separate RNA extraction and qPCR reaction for each sample with a fixed cost and reaction time. While automation and improved logistics can increase the capacity of these tests, they cannot exceed this lower bound dictated by one extraction and reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information on viral strains and host immune response.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, </span><span style = "font-weight:bold;">ApharSeq</span><span> (</span><span style = "font-weight:bold;">A</span><span>mplicon </span><span style = "font-weight:bold;">P</span><span>ooling by </span><span style = "font-weight:bold;">H</span><span>ybridization </span><span style = "font-weight:bold;">A</span><span>nd </span><span style = "font-weight:bold;">R</span><span>NA-</span><span style = "font-weight:bold;">S</span><span>eq), includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. Considering these improvements, our proposed workflow is estimated to reduce costs by 10-50 fold, labor by 5-100 fold, automated liquid handling by 5-10 fold, and reagent requirements by 100-1000 fold compared to existing methods. </span></div></div></div>

[STEPS]
?. [Apharseq]
Prepare poly dT beadsUse 5 µl poly dT beads/sampleWash beads twice in binding buffer:      2.1 Resuspend in binding buffer      2.2 Magnetize and remove buffer 3.  Resuspend beads in 320 µl binding buffer
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Hybridization to beadsAdd 320 µl inactivated viral sample to 320 µl beads in binding bufferIncubate   at    while mixingMagnetize and remove supernatantIncubate  at   while mixing
0 Room temperature
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Primer annealing1. Resuspend beads in 50 µl 1:1 mix of binding buffer and 10 µM RT primer(s)2. Heat sample to  for   then incubate  for at least 2. Incubate samples   at  . Mix by rotating or by pipetting every 5 minutes 3. Magnetize beads and discard supernatant
72 °C
on ice
0 Room temperature
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Wash and Pool1. Resuspend beads in 450 µl wash buffer A2. Magnetize and remove 380 µl  2. Resuspend beads in remaining 70µl and pool samples.3. Wash beads twice in wash buffer B:    2.1 Resuspend in 500 µl buffer B    2.2 Magnetize and carfully remove buffer
Pool can be stored in RNA later at    for a week. If stored,  prior to step 5, wash twice in Wash buffer B
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Reverse Transcription      1.1 Resuspend in 50 µl 1xRT buffer      1.2 Magnetize and remove buffer 2.  Resuspend in 50 µl RT reaction mix  3.  Incubate
25 °C
42 °C
70 °C
We also found a standard RT-PCR kit to work on the beads (PrimeScript™ One Step RT-PCR). Importantly we execute the RT part of the reaction, and only then add the PCR primers.
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cDNA elution1. Incubate the beads for   at 2. Magnetize and transfer the supenatant immediately to a new tube
98 °C
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Cleanup by 2x SPRI  1. Add 100 µl SPRI beads and mix well2. Incubate 3. Magnetize: keep on magnet for   and remove supernatant4. Keep on magnet and wash twice with 70% ethanol      4.1 Add 100 µl 70% ethanol     4.2 Incubate      4.3 Remove the ethanol without disturbing the beads5. Air dry to remove traces of ethanol 6. Resuspend in 23 µl 10 mM Tris 8.07. Incubate 8. Magnetzie and transfer 21 µl to new tube
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Library PCR1. Add to each sample pool25 µl Kapa RM 2 µl 10 µM PCR-primer F2 µl 10 µM PCR-primer R2. PCR program:98°C 3 minutes30 x {95°C 20sec, 59°C 15sec, 72°C 50sec};72°C 10 min10°C2. PCR program:98°C 3 minutes30 x {95°C 20sec, 59°C 15sec, 72°C 50sec};72°C 10 min10°C
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Cleanup by 1x SPRI (see step 8)Elute in 15 µl 10 mM Tris 8.0
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Library assessment1. Determine library concentration (e.g. by Qubit) and size (by tape/gel)2. If necessary, clean primer dimers by running on gel or by 1x SPRI
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SequenceOn an Illumina machine. Assign 5 million reads per 100 sample pool.Assign at least 20 cycles to R1 (barcode + UMI) and either additional 30 cycles to R1 or 30 cycles to R2
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Data Analasys Outline1. Demultiplex pool indices with the bcl2fastq script (Illumina)2. Process each fastq file independently     2.1 Split reads based on RT primer target (E/N1/ActB) and barcode (R1:4-8 + R1:13-17, inclusive)     2.2 Filter reads that do not exhibit their expected sequence                i.e. read with E primer should have the E sequence immediately after the primer     2.2 Collect UMI sequence per read (R1:1-4 + R1:9-12 + R1:18-19)     2.3 Cluster UMI sequences (e.g. by hamming distance and/or with UMI-tools)     2.4 Report unique molecules per target/sample
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Proteinase K (PK) treatmentWash with 500 µl PK bufferResuspend in 500 µl PK mix (0.4mg/ml PK in PK bufer)Incubate     at   while rotating.Magnetize and wash three times in 1ml wash buffer A Magnetize and wash twice in 1ml wash buffer B Incubate    at  while rotating.Magnetize and wash three times in 1ml wash buffer A Magnetize and wash twice in 1ml wash buffer B
0 Room temperature
This step may need calibration to shorten incubation time at RT or 37C