TITLE: TGen North high throughput SARS-CoV-2 tiled amplicon sequencing
AUTHORS: Heather Centner, Ashlyn Pfeiffer, Tporter , Ronuck Patel, Andrew Goedderz, Timothy McDaniel, Elizabeth Driebe, Dave Engelthaler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Our goal for this project was to incorporate and operationalize a lean laboratory processing method that</div><div class = "text-block">decreases costs, time, and complexity and increases throughput and accessibility for SARS-CoV-2 tiled amplicon sequencing. </div><br/></div>

[STEPS]
?. [Sample selection]
Verified specimen typesKnown SARS-CoV-2 positive clinical specimens, ≥50uL volume (ideally ≥500uL)NP swabs in sterile saline or viral transport mediaAnterior nasal swabs in sterile saline or vital transport mediaSalivaRemnant swabs from rapid antigen test platforms, suspended in sterile saline or viral transport media (such as Abbott ID NOW) **High viral load specimens (≤ Ct 30 on most PCR tests) will yield higher quality data in sequencing workflows. Lower viral load samples may be sequenceable, but often yield less consistent/lower quality results. Additional validation may be necessary to determine a more precise cut-off.
?. [Initial sample processing]
Specimen storage recommendationsUpon receipt of specimens:Refrigerated specimens should be maintained at 2-8°C if they will be processed within 7 days. Otherwise, they should be frozen at -20°C for longer term storage.Specimens that are received frozen should be kept frozen at -20°C until ready to process. They may be thawed at 4C or at room temperatureVery long term storage of specimens (storage upon completion of processing) should occur at -70°C Metadata to record (for research applications, PHI should NOT be recorded)County of sample collectionDate of collectionMetadata to record (for research applications, PHI should NOT be recorded)County of sample collectionDate of collection
?. [Initial sample processing]
Transfer to long term storage vesselIf necessary, transfer sample volume into a smaller, long term storage container (i.e. sterile 2mL screw-cap tube with O-ring)**Use a BSC and appropriate PPE while handling active agent**Both the original specimen and a new 2mL screw-cap tube should be given corresponding labels prior to volume transfer Use a p1000 pipette to transfer up to 1,500uL from the original specimen container to a 2mL screw-cap tube
?. [Abbreviations]
GeneralPPE - Personal Protective EquipmentBSC - Biological Safety CabinetcDNA - complementary DNART - reverse transcriptasePCR - Polymerase Chain ReactionrtPCR - realtime PCRRT-PCR - Reverse Transcriptase PCRrt RT-PCR - realtime Reverse Polymerase PCRCt - Cycle threshold ControlsNEC - Negative Extraction ControlPEC - Positive Extraction ControlControlsNEC - Negative Extraction ControlPEC - Positive Extraction Control
?. [Sample Array and inactivation]
Sample array and inactivationIf applicable, separate specimens into high and low volume processing sets≥50uL = Low volume workflow≥500uL = High volume workflowProceed to appropriate inactivation method below
?. [Sample Purification + DNase treatment]
DNase treatment is a required part of the workflow, as it removes significant amounts of background human DNA from your viral samples. REAGENT PREPARATIONDNase reagent mixture**keep refrigerated when not in use, use within 24 hoursPer plate (96 samples, ~10% extra)525uL 5uL DNase I Enzyme525uL DNase I Reaction Buffer4.2mL Molecular Grade Water20% PEG, 2.5M NaCl solution**Store at room temperature, use within 1 month**You will use ~5mL per plate of 96 samples200mL 50% PEG 8000250mL 5M NaCl8.5mL 1X Tris EDTA5Bring total volume to 500mL with Molecular Grade Water70% EtOH**store at room temperature, use within 1 week**you will use ~45mL per plate of 96 samples35mL 100% Ethanol15mL Molecular Grade Water20% PEG, 2.5M NaCl solution**Store at room temperature, use within 1 month**You will use ~5mL per plate of 96 samples200mL 50% PEG 8000250mL 5M NaCl8.5mL 1X Tris EDTA5Bring total volume to 500mL with Molecular Grade Water70% EtOH**store at room temperature, use within 1 week**you will use ~45mL per plate of 96 samples35mL 100% Ethanol15mL Molecular Grade Water70% EtOH**store at room temperature, use within 1 week**you will use ~45mL per plate of 96 samples35mL 100% Ethanol15mL Molecular Grade WaterProceed to appropriate protocol below
?. [Sample qualification]
Establishing an early quality measureIt is advantageous to define a pre-library prep quality measure that is predictive of successful sequencing. Viral load is recommended. TGen has determined that a Ct value of ≤34 on a SARS-CoV-2 N gene target RT-PCR assay is predictive of a high quality genome. However, validation may yield a different cutoff in a different lab. A predictive cutoff can be established by plotting pre-library prep viral load quantities against their resulting genome quality measures. A drop-off in genome quality should be apparent at some point where viral load is lower. Using cutoffs efficientlyIf a large percentage (>20%) of samples are expected to be low viral load and thus not expected to yield quality data, it is advisable to remove low viral load samples from the workflow at this point. This will prevent wasted reagent and labor that would occur from proceeding with these poor quality samples. If ≥80% of samples are expected to yield high quality data,  the labor investment in removing the poor quality samples surpasses the reagent/labor waste incurred by processing them. For this reason, it is advised not to remove individual samples from the larger set.
?. [cDNA synthesis]
NEB LunaScript RT SuperMix KitCombine the following in a 96-well thermal cycler compatible microplate **The following is a validated partial reaction (50% decrease from NEB recommended volumes)2uL LunaScript RT SuperMix 5X           - 96-well plates containing 2uL SuperMix per well may be prepared ahead of time, sealed, and stored at -20C8uL RNA (post-DNase treatment/purification product)MixPlate vortexer ≥850rpm for ≥30 secondsAlternatively, pipette mix 3-4 times with every addition of 8uL RNA          - 96-well plates containing 2uL SuperMix per well may be prepared ahead of time, sealed, and stored at -20C8uL RNA (post-DNase treatment/purification product)MixPlate vortexer ≥850rpm for ≥30 secondsAlternatively, pipette mix 3-4 times with every addition of 8uL RNAMixPlate vortexer ≥850rpm for ≥30 secondsAlternatively, pipette mix 3-4 times with every addition of 8uL RNACentrifuge brieflyThermal cycler program:Heated Lid:105CVolume: 10uLCYCLE STEP 	    TEMPERATURE       TIME 	        CYCLES 1. Primer Annealing      25°C                  2 minutes              12. cDNA Synthesis        55°C                  10 minutes            13. Heat Inactivation 	 95°C                  1 minute                1       4. Hold                            10°C                   ∞
?. [ARTIC V4 PCR]
ARTIC V4 Primer Panel InfoPrimer Design and pooling concentrations available on GitHub https://github.com/artic-network/artic-ncov2019/tree/master/primer_schemes/nCoV-2019/V4Primer sequences: https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V4/SARS-CoV-2.primer.bedPooling instructions: https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V4/README20x mix instructions:Pool 100uM stocks of primers according to GitHub README instructionsDilute pools by a factor of 2.62158108 (1 part primers, 2.62 parts 1x Tris EDTA)Resulting concentrations in 20x primer mix17, 27, 30, 61, 74 (5x volume) = 1500nM13, 45, 79 (2x volume) = 600nMEverything else (1x volume) = 300nMResulting concentrations in 20x primer mix17, 27, 30, 61, 74 (5x volume) = 1500nM13, 45, 79 (2x volume) = 600nMEverything else (1x volume) = 300nM
?. [PlexWell 384 protocol]
Pooling PCR productFor each sample, combine 25uL ARTIC pool 1 product with 25uL ARTIC pool 2 product (entire volumes). Perform a 1:9 dilution on the pool 1/pool 2 combination (i.e. 5uL pooled product, 45uL molecular grade water)
?. [ARTIC V4 PCR]
Tiled amplicon PCR instructionsPrepare sufficient volume of mastermix for the number of samples being processed, plus 10% extra for pipette error. This mastermix will need to be created twice, once for primer pool 1 and again for primer pool 2. MasterMix calculations, per sampleMolecular Grade Water    8.75 uLPrimer Mix (20x)               1.25 uLQ5                                        12.5 uLMasterMix calculations, plate of 96 plus 10%Molecular Grade Water    924 uLPrimer Mix (20x)               132 uLQ5                                        1320 uLMasterMix calculations, plate of 96 plus 10%Molecular Grade Water    924 uLPrimer Mix (20x)               132 uLQ5                                        1320 uL
?. [Control Generation]
NEC (Negative Extraction Control)Negative media (sterile saline or viral transport media)PEC (Positive Extraction Control)Known concentration of SARS-CoV-2 RNA in a clinical matrixConcentration: at or near lower limit of viral input (Ct ~32)Clinical matrix: remnant clinical specimens known to be negative for SARS-CoV-2Consider using a strain that is not common in the populations being sequenced (i.e. a basal strain) PEC (Positive Extraction Control)Known concentration of SARS-CoV-2 RNA in a clinical matrixConcentration: at or near lower limit of viral input (Ct ~32)Clinical matrix: remnant clinical specimens known to be negative for SARS-CoV-2Consider using a strain that is not common in the populations being sequenced (i.e. a basal strain)
?. 
?. [Sample Purification and DNase treatment]
DNase treatment is a required part of the workflow, as it removes significant amounts of background human DNA from your viral samples. REAGENT PREPARATIONDNase reagent mixture**keep refrigerated when not in use, use within 24 hoursPer plate (96 samples, 10% extra)528uL 5uL DNase I Enzyme528uL DNase I Reaction Buffer4,224uL Molecular Grade Water
20% PEG, 2.5M NaCl solution**Store at room temperature, use within 1 month**You will use ~5mL per plate of 96 samples250mL 50% PEG 8000312.5mL 5M NaCl10.6mL 1X Tris EDTA51.9mL Molecular Grade Water
70% EtOH**store at room temperature, use within 1 week**you will use ~45mL per plate of 96 samples35mL 100% Ethanol15mL Molecular Grade Water20% PEG, 2.5M NaCl solution**Store at room temperature, use within 1 month**You will use ~5mL per plate of 96 samples250mL 50% PEG 8000312.5mL 5M NaCl10.6mL 1X Tris EDTA51.9mL Molecular Grade Water
70% EtOH**store at room temperature, use within 1 week**you will use ~45mL per plate of 96 samples35mL 100% Ethanol15mL Molecular Grade Water70% EtOH**store at room temperature, use within 1 week**you will use ~45mL per plate of 96 samples35mL 100% Ethanol15mL Molecular Grade Water
?. [PlexWell 384 protocol]
Proceed to PlexWell protocol:
?. [Load on Illumina Sequencer]
Sequencing coverage recommendationsTGen routinely uses a NextSeq Mid Output 300 cycle kit, with up to 576 samples per run. This gives ~65 megabases of sequencing data to each sample. More samples per run may be independently validated by gradually increasing cluster density. Run quality metrics should be closely monitored, and increases in cluster density should be halted when metrics show a marked decline or go out of spec. As long as ≥65Mb per sample is achieved, scaling to smaller or larger sequencing kits is possible.
?. [ARTIC V4 PCR]
PCR Assay AssemblyCombine 22.5uL Master Mix2.5uL cDNAMixPlate vortexer ≥850rpm for ≥30 secondsAlternatively, pipette mix 3-4 times with every addition of 8uL RNAMixPlate vortexer ≥850rpm for ≥30 secondsAlternatively, pipette mix 3-4 times with every addition of 8uL RNACentrifuge BrieflyThermal cycler program:Heated Lid:105CVolume: 25uLCYCLE STEP 	    TEMPERATURE 	    TIME 	        CYCLES 1. Heat Activation         98°C                           30 sec            12. Denaturation             98°C                           15 sec             3. Annealing       	         65°C                           5 mins.           Repeat steps 2-3 for a total of 35 cycles 4. Hold                            10°C                            ∞