TITLE: CLIAHUB Automated RNA Extraction & RT-PCR Protocol V2
AUTHORS: Amy Lyden, Emily Crawford, Vida Ahyong, Manu Vanaerschot, Paula Hayakawa Serpa, Preethi Raghavan, Joana Cabrera, Michael Borja, Spyros Darmanis, Eric Chow, Joseph Derisi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Github: (to be added)</div><div class = "text-block">Laboratory testing for COVID-19 infection is an important part of both the individual patient care and public health responses to this emerging outbreak.  Results are used to guide containment efforts, including isolation and contact tracing, and make clinical diagnosis for supportive management and experimental therapies (there is no known efficacious treatment available for COVID-19 infection as of March 2020).  Real-time reverse transcriptase PCR (rRT-PCR) testing is a well-established method to detect viral RNA in clinical samples, and several labs around the world have designed and validated primers and probes for this purpose that are also used in this assay (4 and 5). </div><div class = "text-block">This assay is intended to qualitatively detect COVID-19 viral RNA in patient samples to enable the diagnosis of COVID-19 disease.  Testing should be performed for patients with signs and symptoms of potential COVID-19 infection.  Positive results would generally indicate active infection but do not rule out co-infection with other viruses, bacteria or other pathogens.  Negative test results do not absolutely rule out infection, and results should be interpreted in the clinical context. </div><div class = "text-block">The oligonucleotide primers and probes for detection of SARS-CoV-2 were selected from regions of the Nucleoprotein (N gene)and the Envelope protein (E gene). An additional primer/probe set to detect the human RNase P gene (RP) in control samples and clinical specimens is also included in the panel. </div><div class = "text-block">RNA isolated and purified from upper respiratory specimens is reverse transcribed to cDNA and subsequently amplified on the Bio-Rad CFX Real Time PCR Machine. In the process, the probes anneal to a specific target sequence located between the forward and reverse primers.  During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal.  With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity.</div><div class = "text-block">Detection of viral RNA not only aids in the diagnosis of illness but also provides epidemiological and surveillance information.</div></div>

[STEPS]
?. [Reagent Plate Preparation]
All reagent plates should be barcoded. Record barcode of the plate and lot number of the reagents used in each plate (ie record the lot numbers of DNAse, DNAse Digestion Buffer and water used to make DNAse master mix). It is also recommended that you label the reagents with a human readable label and number the reagents to faciliate matching with the automation pipeline on the Bravo.
?. [Reagent Plate Preparation]
Zymo DNA/RNA Shield - Add 500uL of 2x Zymo DNA/RNA Shield into 2mL deep well plates (for addition of samples not collected in shield) in a PCR Workstation. Aliquot using the Thermo Multidrop liquid dispenser. Seal with ThermoFisher blue rubber seals and store at room temperature.
?. [Reagent Plate Preparation]
Proteinase K - Add 1.2 ml Proteinase K Storage Buffer to reconstitute per 20 mg lyophilized Proteinase K to make a 20mg/mL stock. Aliquot 3uL Proteinase K at 20mg/mL, using the Integra Viaflo 96 into a 1mL 96 deep well plate. Seal and store at -20C, up to a week prior to nucleic acid extraction.
?. [Reagent Plate Preparation]
Viral DNA/RNA Buffer - Add 1 ml beta-mercaptoethanol (BME) per 200 ml Viral DNA/RNA Buffer, final concentration 0.5% (v/v). Aliquot 450uL of Viral DNA/RNA Buffer with BME using the Thermo Multidrop in 1mL 96 deep well plate Seal and store at RT protected from light, up to a week prior to nucleic acid extraction.
?. [Reagent Plate Preparation]
Magbeads - Vortex MagBeads and aliquot into a 1mL deep well source plate. Mix with Integra Viaflo 96. Aliquot 30uL using the Integra Viaflo 96 into Bio-Rad 96 PCR plate. You must mix the MagBeads between each aliquot to avoid settling. Seal and store upside down to avoid settling, at room temperature up to a week prior to nucleic acid extraction.
?. [Reagent Plate Preparation]
MagBead DNA/RNA Wash 1 & 2 - Add 80mL of isopropanol to the MagBead DNA/RNA Wash 1 concentrate. Add 120mL of isopropanol to the MagBead DNA/RNA Wash 2 concentrate. Aliquot 550uL of DNA/RNA Magbead Wash Buffers 1 and 2 using the Thermo Multidrop into 1mL 96 deep well plate. Heat seal and store at room temperature in a flammables cabinet, up to a week prior to nucleic acid extraction.
?. [Reagent Plate Preparation]
Ethanol - Aliquot 1.1mL of 100% ethanol, using the Thermo Multidrop, into a 1mL 96 deep well plate plate. Heat seal and store at room temperature in a flammables cabinet, up to a week prior to nucleic acid extraction.
?. [Reagent Plate Preparation]
DNAse I - Add 275 µl DNase/RNase-Free Water to reconstitute the lyophilized DNase I at 1 U/µl. Mix by gentle inversion. Aliquot 55uL of DNAse I in DNAse Digestion buffer, using the Thermo Multidrop in Bio-Rad 96 PCR plate. Seal and store at 4C for for up to a week.
?. [Reagent Plate Preparation]
RNA Prep Buffer - Aliquot 550uL of RNA Prep Buffer using the Thermo Multidrop into 1mL 96 deep well plate. Heat seal and store at room temperature, up to a week prior to nucleic acid extraction.
?. [Reagent Plate Preparation]
Water - Aliquot 100uL of DNAse/RNAse free water using the Thermo Multidrop into Bio-Rad 96 PCR plate, sealed and stored at room temperature, up to a week prior to nucleic acid extraction
?. [Reagent Plate Preparation]
Human RNA extraction control (HRC) - Prepare a 1x PBS solution from the 10x stock solution using nuclease-free water. Prepare 1:1 PBS:Shield solution by diluting 1x PBS in 2x Shield. Dilute extracted HeLa RNA 1 in 1000 in PBS:Shield to a final concentration of 120 ng/ml. Mix by inverting tube and spin down to remove bubbles. Prepare aliquots (2x 500 μl per plate needed), store at 4C for up to 7 days.
?. [Reagent Plate Preparation]
Positive control (PC): Dilute high titer patient sample (10^7 cp/ml, already in Shield) to 10^5 cp/ml in HRC (see above). Mix by inverting tube and spin down to remove bubbles. Prepare aliquots (2x 500 μl per plate needed), store at 4C for up to 7 days.
?. [Reagent Plate Preparation]
PBS: Prepare 1x PBS solution from the 10x stock solution using nuclease-free water
?. [Reagent Plate Preparation]
NTC: Use nuclease-free water as NTC.
?. [qPCR MM Preparation]
Preparation of primer/probe mixes - the primer/probe mix will be diluted 1/20 in the final reaction (0.5 μl of primer/probe mix per 10 μl reaction). Primer/probe mixes are prepared as follows:   ABCDEF1E-gene primer/probe mixStock concentration
  (μM)Desired final concentration in
  reaction (nM)Required concentration in
  primer/probe mix (nM)Dilution factor from stockVolume for 1 reaction
  (μl)2SARS-CoV-2_E_Fwd2004008000250.023SARS-CoV-2_E_Rev2004008000250.024SARS-CoV-2_E_Prb-FAM1002004000250.025Nuclease-free water ---0.446Total (μl)    0.5  ABCDEF1N-gene primer/probe mixStock concentration
  (μM)Desired final concentration in
  reaction (nM)Required concentration in
  primer/probe mix (nM)Dilution factor from stockVolume for 1 reaction
  (μl)2NIID_2019-nCOV_N_F220050010000200.0253NIID_2019-nCOV_N_R220050010000200.0254NIID_2019-nCOV_N_P21002004000250.025Nuclease-free water ---0.436Total (μl)    0.5  ABCDEF1RNaseP primer/probe mixStock concentration
  (μM)Desired final concentration in
  reaction (nM)Required concentration in
  primer/probe mix (nM)Dilution factor from stockVolume for 1 reaction
  (μl)2Rnase P Fwd2002004000500.013Rnase P Rev2002004000500.014RNAseP Probe HEX1005010001000.0055Nuclease-free water ---0.4756Total (μl)    0.5
ABCDEF1E-gene primer/probe mixStock concentration
  (μM)Desired final concentration in
  reaction (nM)Required concentration in
  primer/probe mix (nM)Dilution factor from stockVolume for 1 reaction
  (μl)2SARS-CoV-2_E_Fwd2004008000250.023SARS-CoV-2_E_Rev2004008000250.024SARS-CoV-2_E_Prb-FAM1002004000250.025Nuclease-free water ---0.446Total (μl)    0.5
ABCDEF1N-gene primer/probe mixStock concentration
  (μM)Desired final concentration in
  reaction (nM)Required concentration in
  primer/probe mix (nM)Dilution factor from stockVolume for 1 reaction
  (μl)2NIID_2019-nCOV_N_F220050010000200.0253NIID_2019-nCOV_N_R220050010000200.0254NIID_2019-nCOV_N_P21002004000250.025Nuclease-free water ---0.436Total (μl)    0.5
ABCDEF1RNaseP primer/probe mixStock concentration
  (μM)Desired final concentration in
  reaction (nM)Required concentration in
  primer/probe mix (nM)Dilution factor from stockVolume for 1 reaction
  (μl)2Rnase P Fwd2002004000500.013Rnase P Rev2002004000500.014RNAseP Probe HEX1005010001000.0055Nuclease-free water ---0.4756Total (μl)    0.5
?. [qPCR MM Preparation]
Preparation of mastermix - one plate will contain 96 individual reactions of 10 μl for one primer/probe set (960 μl), but an additional 20 μl are prepared to ensure enough material is available for plating (total of 980μl). The full composition of the qPCR reaction is as follows (note that sample is not added at this point). INSERT TABLE  A1  ABCDEF1Per reactionPer plate2Number of reactions1 Number of plates13Luna Universal Probe 1step qPCR mix (2x)5 Luna Universal Probe 1step qPCR mix (2x)4904Luna WarmStart RT Enzyme0.5 Luna WarmStart RT Enzyme495Primer/probe mix (μl)0.5 Primer/probe mix (μl)496Sample (μl)4 Sample (μl)3927Total (μl)10 Tota l(μl)9808   Volume without sample588
A1
ABCDEF1Per reactionPer plate2Number of reactions1 Number of plates13Luna Universal Probe 1step qPCR mix (2x)5 Luna Universal Probe 1step qPCR mix (2x)4904Luna WarmStart RT Enzyme0.5 Luna WarmStart RT Enzyme495Primer/probe mix (μl)0.5 Primer/probe mix (μl)496Sample (μl)4 Sample (μl)3927Total (μl)10 Tota l(μl)9808   Volume without sample588
?. [qPCR MM Preparation]
Filling qPCR plates with mastermix (6 μl/well) -	The mastermix is plated in Bio-Rad 384 well PCR plates using the Dragonfly (6 μl per well) as follows:
?. [qPCR MM Preparation]
Freezing qPCR plates at -20C - spin down plate(s) at 1000 rpm for 1 min, cover plate(s) using F foil and store plate(s) at -20C until use.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Decontaminate the workstation - spray down the area and all surfaces with RNaseZAP and wipe down. Spray down the area and all surfaces with 70% ethanol and wipe down.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Take out Proteinase K from -20C. Allow it to thaw prior to starting extraction.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Gather all other reagents EXCEPT the DNase I plate and qPCR MM plate. Inspect the reagent plates to make sure they appear to have equal volumes in each well.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Pick up an empty sealed barcoded Full skirt Bio-Rad PCR plate for RNA
?. [Preparing for RNA Extraction on the Agilent Bravo]
Spin down (1 minute at 215 rcf) all plates EXCEPT the RNA and Magbead plates. Note: when prompted, spin down DNase I and qPCR plate with the same centrifugation settings.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Organize plates in the order they'll be used throughout the extraction.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Spin down the sample plate (stored at 4C). Leave sealed until everything else is ready.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Turn on the Agilent Bravo instrument and open the VWorks software.
?. [Preparing for RNA Extraction on the Agilent Bravo]
Open up the RNA extraction form which links to the protocol.Example VWorks RNA Extraction Form
?. [Preparing for RNA Extraction on the Agilent Bravo]
?. [Preparing for RNA Extraction on the Agilent Bravo]
Press "Run Protocol" on the form file.
?. [Preparing for RNA Extraction on the Agilent Bravo]
During the protocol, follow the prompts to unseal reagent plates and take out reagent plates from the freezer at the appropriate time. Be sure to dispose of reagent plates, liquid waste, and tips in the appropriate chemical waste bins.
?. [RNA Extraction on the Agilent Bravo]
The Bravo transfers 180uL of stock sample into a 1ml deep well plate with pre-aliquoted Proteinase K on Deck 5.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 360uL (from deck 9) of the mix per well from a prepared stock plate and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The Bravo mixes the Magbeads (deck 6).
?. [RNA Extraction on the Agilent Bravo]
It aspirates 20uL of beads (deck 6) to be mixed with the sample in Proteinase K + Viral Buffer Mix (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The beads and sample are mixed (deck 5) for 10mins.
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 5mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is swapped with Magbead DNA/RNA Wash Buffer 1.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 500uL (from deck 9) of the Wash 1 Buffer and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 5mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is swapped with Magbead DNA/RNA Wash Buffer 2.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 500uL (from deck 9) of the Wash 2 Buffer and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 5mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is swapped with 100% ethanol - enough for two washes.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 500uL (from deck 9) of the ethanol and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 2mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is currently 100% ethanol.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 500uL (from deck 9) of the ethanol and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 2mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is swapped with DNase Mix (DNase + Digestion buffer + water)
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 50uL (from deck 9) of the DNase mix and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate incubates (deck 5) for 15mins at room temperature.
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is swapped with RNA Prep Buffer.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 500uL (from deck 9) of the RNA Prep Buffer and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is mixed (deck 5) for 10mins.
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 5mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is swapped with 100% ethanol - enough for two washes.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 500uL (from deck 9) of the ethanol and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 2mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is currently 100% ethanol.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 500uL (from deck 9) of the ethanol and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 2mins.
?. [RNA Extraction on the Agilent Bravo]
The supernatant (deck 7) is removed to waste (deck 1).
?. [RNA Extraction on the Agilent Bravo]
The reagent plate (deck 9) is swapped with 100% nuclease-free water.
?. [RNA Extraction on the Agilent Bravo]
The Bravo picks up 45uL (from deck 9) of water and mixes it with the samples (deck 5).
?. [RNA Extraction on the Agilent Bravo]
The plate is moved to a magnet (deck 7) to incubate for 5mins.
?. [RNA Extraction on the Agilent Bravo]
The RNA eluted in water (deck 7) is collected and dispensed into a final plate (deck 3).
?. [RNA Extraction on the Agilent Bravo]
Spin down the plate (1 minute at 215 rcf) and immediately proceed to transferring your extracted RNA into a pre-prepared 384-well qPCR plate.
?. [Setting up qPCR]
Clean off Bravo deck.
?. [Setting up qPCR]
Open qPCR plating VWorks form, and set up deck according to the form - including adding your spun down RNA plate from the extraction protocol onto Deck 6 and the thawed and spun down qPCR MM plate on Deck 9.Example qPCR plating form in VWorks Software
?. [Setting up qPCR]
Press "Run protocol".
?. [Setting up qPCR]
The Bravo will aliquot 4uL of RNA each into A1 quadrant, B1 quadrant and A2 quadrant.
?. [Setting up qPCR]
Seal qPCR plate with Biorad B optical seal. Do not touch the top of the seal to avoid leaving marks. Remove the tabs.
?. [Setting up qPCR]
Spin down qPCR plate for 1 minute at 215 rcf.
?. [Setting up qPCR]
Run qPCR protocol on BioRad CFX thermocycler. Ensure detection for both FAM and HEX channels.   ABCDEF1StepStage# CyclesTemperatureTime2RT1155 oC10 min3Initial denaturation2195 oC1 min4Amplification34595 oC10 seconds558 oC30 seconds
ABCDEF1StepStage# CyclesTemperatureTime2RT1155 oC10 min3Initial denaturation2195 oC1 min4Amplification34595 oC10 seconds558 oC30 seconds
?. [Setting up qPCR]
Freeze RNA at -80C.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Pipette transfer for shield samples only - perform in a PCR or BSC hood
?. [qPCR MM Preparation]
N-gene primers and probe (SARS-CoV-2) - manufactured by IDT in template-free facility  ABC1Primer/probe namePrimer/probe sequenceFluorophore2NIID_2019-nCOV_N_F2AAATTTTGGGGACCAGGAAC-3NIID_2019-nCOV_N_R2TGGCACCTGTGTAGGTCAAC-4NIID_2019-nCOV_N_P2-FAMATGTCGCGCATTGGCATGGAFAM
ABC1Primer/probe namePrimer/probe sequenceFluorophore2NIID_2019-nCOV_N_F2AAATTTTGGGGACCAGGAAC-3NIID_2019-nCOV_N_R2TGGCACCTGTGTAGGTCAAC-4NIID_2019-nCOV_N_P2-FAMATGTCGCGCATTGGCATGGAFAM
?. [qPCR MM Preparation]
E-gene primers and probe (SARS-CoV-2) - manufactured by IDT in template-free facility  ABC1Primer/probe namePrimer/probe sequenceFluorophore2SARS-CoV-2_E_FwdACAGGTACGTTAATAGTTAATAGCGT-3SARS-CoV-2_E_RevATATTGCAGCAGTACGCACACA-4SARS-CoV-2_E_Prb-FAMACACTAGCCATCCTTACTGCGCTTCGFAM
ABC1Primer/probe namePrimer/probe sequenceFluorophore2SARS-CoV-2_E_FwdACAGGTACGTTAATAGTTAATAGCGT-3SARS-CoV-2_E_RevATATTGCAGCAGTACGCACACA-4SARS-CoV-2_E_Prb-FAMACACTAGCCATCCTTACTGCGCTTCGFAM
?. [qPCR MM Preparation]
RNAseP primers and probe (human control) - manufactured by IDT in template-free facility  ABC1Primer/probe namePrimer/probe sequenceFluorophore2RNAse P ForwardAGATTTGGACCTGCGAGCG-3RNAse P ReverseGAGCGGCTGTCTCCACAAGT-4RNAse P P-HEXTTCTGACCTGAAGGCTCTGCGCGHEX
ABC1Primer/probe namePrimer/probe sequenceFluorophore2RNAse P ForwardAGATTTGGACCTGCGAGCG-3RNAse P ReverseGAGCGGCTGTCTCCACAAGT-4RNAse P P-HEXTTCTGACCTGAAGGCTCTGCGCGHEX
?. [Interpreting Results]
Positive control - PC reaction should produce a positive result with a Ct value within the following expected ranges: N gene
?. [Interpreting Results]
Negative controls - NTC should be negative and exhibit no fluorescence growth curves that cross the threshold line. If a false positive occurs with one or more of the primer and probe NTC reactions, sample contamination may have occurred. PBS should be negative and exhibit no fluorescence growth curves that cross the threshold line. If a false positive occurs with one or more of the primer and probe PBS or NTC reactions, sample contamination may have occurred.
?. [Interpreting Results]
RNAse P should be positive at Ct ≤38 for all negative clinical samples and HRC. Acceptable results for the RP in patient samples indicates the presence of sufficient nucleic acid from the human RNase P gene, and thus, acceptable specimen quality. Failure of RP detection in HRC may indicate improper assay set up or instrument malfunction. Failure of RP in patient samples but detection in HRC may indicate extraction failure, PCR inhibition, or absence of sufficient human cellular material. Samples that are positive for viral detection do not require amplification of RP target to be valid
?. [Interpreting Results]
Human RNA Control - HRC should be negative for SARS-CoV specific primer/probe sets. A fluorescence growth curve for N gene or E gene that crosses the threshold line may indicate contamination of reagents or cross contamination of samples. If the HRC exhibits a positive signal for Coronavirus specific primer/probe sets, the run is invalid.
?. [Interpreting Results]
Patient samples  ABCDE1NERPInterpretationAction2PosPosPos or NegDetectedReport as Detected31 of 2 pos, or 1 pos with Ct value > 38.0Pos or NegIndeterminateRepeat with new extraction; if same result Report as Indeterminate4NegNegPosNot DetectedReport as Not Detected5NegNegNegInvalidRepeat with new extraction; if same result Report as Invalid
ABCDE1NERPInterpretationAction2PosPosPos or NegDetectedReport as Detected31 of 2 pos, or 1 pos with Ct value > 38.0Pos or NegIndeterminateRepeat with new extraction; if same result Report as Indeterminate4NegNegPosNot DetectedReport as Not Detected5NegNegNegInvalidRepeat with new extraction; if same result Report as Invalid
?. [Interpreting Results]
Bring any unexpected results to the supervisor or section director before sending the result file for reporting. a. Any unexpected control result. b. Cluster of positive results on a plate map with 2 or more adjacent positive wells. c. Plates with more than 4 Indeterminate or Invalid results. d. Plates with more than 10 positive results.
?. [Interpreting Results]
Negative: a specimen is negative if none of the SARS-CoV-2 markers (N gene, E gene) exhibit growth curves that cross the threshold AND RNase P (RP) is positive (DOES cross the threshold line).
?. [Interpreting Results]
Positive: a specimen is positive for SARS-CoV-2 if both markers (N gene, E gene) exhibit growth curves that cross the threshold line with Cp value
?. [Interpreting Results]
Indeterminate: If only one of the SARS-CoV-2 markers is positive or if both are positive but one or both have Ct > 40.0, the result is inconclusive for SARS-CoV-2 and should be repeated. If repeat is the same, report as Indeterminate.
?. [Interpreting Results]
Invalid: If all targets (N gene, E gene, RNAse P) do not cross the threshold line, the result is invalid and should be repeated. If repeat is the same, report as Invalid.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Transfer 600uL of appropriate controls in the layout outlined in the image below into a 96-well 2mL deep well plate.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Carefully remove swab from samples and dispose in biohazard. Tweezers, upside-down pipette tips and protective gauze may all be useful in this process. Change gloves if sample gets on it.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Pipette transfer for non-shield samples (PERFORM IN APPROPRIATE BSL SPACE IN BSC HOOD)
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Grab pre-prepared plate with 500uL of 2X DNA/RNA shield. Remove 500uL of shield from wells which should have controls (see step 22.1). Add 600uL of indicated control.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Transfer 500uL of up to 84 samples into the remaining empty wells, recording barcodes and well location during sample addition. Pipette well to mix with 2X DNA/RNA shield. Avoid cross-contamination at this step and inspect samples for high viscosity, snot or other irregularities which may lead to difficulties liquid handling.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Sample transfer via Hamilton - the following steps will refer to operation of a Hamilton protocol to transfer samples in tubes with at least 2mL of volume to a 96 well 2mL deep well plate. Custom pathing was implemented to ensure that pipette tips never passed over original tubes. In addition, samples are transferred in groups of 8, aligned in a single column. Pipette tips approach from the top of the plate, to try to contain any potential contamination to a column-wise fashion.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Decontaminate the workstation - Spray down the area and all surfaces with RNAseZap and wipe down. Spray down the area and all surfaces with 70% ethanol and wipe down.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Turn on computer and turn on Hamilton robot. Open up ‘Sample Reformatting’ shortcut on the desktop
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Press the play button on top to open up Hamilton GUI.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Select site and enter name under “User name” and press “continue”
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Ensure the following on the GUI: Correct number of patient samples is entered (this does not include control tubes) If samples will be recapped choose “Recapping - With Custom Pathing”. If samples are not going to be recapped choose “No Recapping - No Custom Pathing” Check “Load carrier and scan barcodes on labware” to ensure that the labware is barcoded and a csv file is generated. If you are transferring to only one deep well plate, turn on checkbox 3 and input 600 uL. If you are transferring to 2 deep well plates, turn on checkbox 3 and 4. If there is at least 2 mL of sample in each tube, enter 600 uL next to boxes 3 and 4. If there is less than 2 mL of sample in each tube, consult your supervisor to determine what volumes to transfer. Select tube type based on inner diameter of sample tube. For tubes with an inner diameter of ~12 mm, choose “small tube”. For tubes with an inner diameter of ~13.5, choose “large tube”. Check “Process transfer from control tubes” to ensure that controls are plated into the deep well.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Load tip rack carrier in lanes 1-6. Load all labware on the autoload tray until you hit the stopper.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Load the tip rack carrier with Hamilton 1000 µL conductive filter tips.  Calculate the number of tips to load by using this formula:  (number of tubes * number of plates) + 16
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Use the GUI to select tip locations based upon the tip boxes you loaded onto the carrier .
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Load tubes into tube rack carriers -	Use tube rack carriers labeled 8, 10, 12, 14, 16, and 18. These numbers correspond to the lanes they should be loaded onto on the Hamilton. Load tubes such that the barcode is visible between the windows of the tube carrier.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Load control tubesNTC: a.	Tube rack carrier 8, pos 1 b.	Tube rack carrier 8, pos 8 c.	Tube rack carrier 18, pos 1 d.	Tube rack carrier 18, pos 8 e.	Tube rack carrier 18, pos 9 f.	Tube rack carrier 18, pos 16 PC: a.	Tube rack carrier 14, pos 9 b.	Tube rack carrier 14, pos 16 HRC: a.	Tube rack carrier 16, pos 1 b.	Tube rack carrier 16, pos 8 PBS: a.	Tube rack carrier 16, pos 9 b.	Tube rack carrier 16, pos 16
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Load the sample tubes into empty slots of the tube rack carrier. Begin with loading the rack on lane 8. Load the tubes in each rack starting at position 1 and ending at position 16. If a position is occupied by a control tube, move onto the next position. Load the remaining racks in the same manner. The racking will result in the columns on a deep well plate as shown in the image below.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Uncap the racked tubes.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Load the racks onto the corresponding lanes on the robot.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Labware loading and barcoding on robot deck Follow the GUI to confirm that the controls are loaded into the correct positions of the tube rack carrier. Tube racks will be automatically loaded and barcoded. Confirm that barcodes are read. If any barcodes raise an error, unload the carrier and use the handheld barcode scanner to input the correct barcode. Follow the GUI to confirm that the deep well plates are on the correct positions of the carrier. The robot will then automatically load the deep well carrier and read the plate barcode.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Once the deep well carrier is loaded, the protocol will begin. Watch the protocol to ensure there is no dripping of sample onto the deck or the plate. Take note of any samples which do drip or do not have a pipette tip lower into the tube - the latter means no sample was transferred.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
After protocol is complete, dispose of waste appropriately and wipe down the deck with 70% ethanol. Seal your sample plate with a blue rubber seal. Recap all tubes.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
A CSV with barcodes for each 2mL deep well plate processed will be exported.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Transfer 600uL of up to 84 samples into the remaining empty wells, recording barcodes and well location during sample addition. Avoid cross-contamination at this step and inspect samples for high viscosity, snot or other irregularities which may lead to difficulties liquid handling.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
Carefully remove swab from samples and dispose in biohazard. Tweezers, upside-down pipette tips and protective gauze may all be useful in this process. Change gloves if sample gets on it.
?. [Transfer Sample Tubes to Plate on the Hamilton or via pipette]
In a hood, carefully remove swab from samples and dispose in biohazard. Tweezers, upside-down pipette tips and protective gauze may all be useful in this process. Change gloves if sample gets on it.