TITLE: AAU-nCoV-2019_Tailed_Long_Amplicon_Sequncing
AUTHORS: Emil Aarre Sorensen, Søren M. Karst, Simon Knutsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the preparation of long read amplicon libraries of nCoV-2019 for sequencing with the Oxford Nanopore MinION platform. The protocol is based on samples of rRNA extracts of the nCoV-2019 virus.</div><div class = "text-block">Sequences for primers can be found under guidelines, currently using V3.1 from CJ020. </div><div class = "text-block"><span>The protocol is based on the ARTIC Networks nCoV-2019 sequencing protocol and modified to our lab at Aalborg Univeristy. </span><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w</span></a></div></div>

[STEPS]
?. [Section 1: Preparations]
Create journal Create a journal with associated unique journal number (CJXXX) inculding metadata for the samples to be processed.Metadata must include reference to a sample registration journal and a sample overview of the samples to be processed. For reference see SOP for setting up journals. Sample preparationsThe protocl describes the workflow as pr 96-well plate basis. A full plate must include 4 negative controls (NFW), a positive control and two empty well for Qubit DNA concentration measurement. Depending on the sample organiziation of the received samples these control samples may be included in varying empty well IDs to minimize any risk of potential mix-ups. (e.g. for a plate with 80 samples, keep the original organiziation and include controls in empty wells).For full sample plates, 7 samples should be skipped to be processed later, preferably in positions C4, F4, C9, F9, F12, G12 and H12 to include the control samples and space for Qubit standards.C4, F4, C9, F9: Negative controls of sterile nuclease free waterF12: Positve control RNA sampleG12 and H12: Empty for Qubit measurement
?. [Section 2: cDNA preparation]
Cleaning pre-PCR laminar flow cabinetShould always be cleaned before and after ues as follows:Wipe down all items in the cabinet; pipettes, tip boxes, trash bin rack, etc. with 70 % ethanol and allow to dry.Wipe down all items in the cabinet; pipettes, tip boxes, trash bin rack, etc. with RNAse awayConsumables for hexamer annealing and cDNA synthesisPlace the following items and reagents in the LAF-bench for UV-treatment (pr plate basis)1 pcs. of skirted PCR-plate - labeled CJXXX_cDNA_pX (journal number and plate number)12 pcs. cap-strips (wipe down plastic bag/container)1 pcs. yellow ice-block (for reagents)1 pcs 96-plate ice-block1 pcs. 8-strip PCR-tubes (for cDNA mastermix aliquots)1 pcs. PCR-strip rack1 pcs of 1.5 mL Eppendorf tube (for cDNA mastermix mixing)1 pcs. reservoir (for mixing of hexamer mastermix)Sufficient amount of each reagent depending on the amount of samples to be processed in the plate (should be calculated in template excel sheets and added to the journal)Ensure sufficient pipette tips are present (wipe new boxes down)Consumables for hexamer annealing and cDNA synthesisPlace the following items and reagents in the LAF-bench for UV-treatment (pr plate basis)1 pcs. of skirted PCR-plate - labeled CJXXX_cDNA_pX (journal number and plate number)12 pcs. cap-strips (wipe down plastic bag/container)1 pcs. yellow ice-block (for reagents)1 pcs 96-plate ice-block1 pcs. 8-strip PCR-tubes (for cDNA mastermix aliquots)1 pcs. PCR-strip rack1 pcs of 1.5 mL Eppendorf tube (for cDNA mastermix mixing)1 pcs. reservoir (for mixing of hexamer mastermix)Sufficient amount of each reagent depending on the amount of samples to be processed in the plate (should be calculated in template excel sheets and added to the journal)Ensure sufficient pipette tips are present (wipe new boxes down)Expose the hood to UV light for 15 minutes.UV sensitive tubes should be wiped down when entering and leaving the cabinet. Gloves should be changed each time entering and leaving the laminar flow cabinet. Production reagents must NEVER be opened outside the laminar flow cabinet.Sheet for calculating Annealing MastermixSheet for calculating cDNA synthesis Mastermix
?. [Section 2: cDNA preparation]
Mastermix for annealing of random hexamers to RNA templateMastermix should be made up in the pre-PCR laminar flow cabinet in the resevoir and aliquoted into the skirted 96 well plate. Mix the following components in a reservoir and alliqout to the 0.2 mL 96 well plate stored in the 96-well ice-block:  ABC1ComponentVolume pr sample96-sample mastermix2Nuclease-free water6 µL619.2 µL350 µM random hexamers1 µL103.2 µL410 mM dNTPs mix (10 mM)1 µL103.2 µL5Total8 µL825.6 µLSeal the plates with strip-caps and note the specific batch number of applied reagents in your journal.
ABC1ComponentVolume pr sample96-sample mastermix2Nuclease-free water6 µL619.2 µL350 µM random hexamers1 µL103.2 µL410 mM dNTPs mix (10 mM)1 µL103.2 µL5Total8 µL825.6 µL
?. [Section 2: cDNA preparation]
Template addition and controlsClean the pre-PCR area and transfer your mastermix plates and RNA samples plates to this area.The pre-PCR area should be cleaned before and after use as follows: Wipe down bench, pipettes, racks, vortex, tubes, etc. in 70 % ethanol and allow to dry Wipe down bench, pipettes, racks, vortex, tubes, etc. with RNAse awaySpin down all template samples to avoid splashing of areosls when handling. Keep the RNA sample plate and cDNA synthesis mastermix plate on ice.Transfer 5 µL template RNA from the sample plate to the mastermix plate containing the annealing mastermix for a total volume of 13 µL in each well. Gently mix by pipetting, seal the plate and spin down to collect liquid at the bottom of the wells. For the negative controls transfer 5 µL of nuclease free water and for the positive control transfer 5 µL of EXT-CJ001-4A.
?. [Section 2: cDNA preparation]
Annealing of hexamersIncubate the plate in PCR-machine with the following program (/protocols/COVID-19/Corona - Random hexamers annealing.js):  AB1TemperatureTime265 °C5 minAfter incubation move samples to ice and leave for at least 1 minute.Random hexamers have now annealed to the template RNA
AB1TemperatureTime265 °C5 min
?. [Section 2: cDNA preparation]
Master mix for cDNA synthesisIn the pre-PCR/master mix cabinet prepare mastermix for cDNA synthesis in a Eppendorf tube as follows and alliqout to an 8-PCR-tube strip 0.2 mL:  ABC1ComponentVolume pr sample96-samples2SSIV Buffer4 µL422.4 µl3100 mM DTT1 µL105.6 µL4RNaseOUT RNase Inhibitor1 µL105.6 µL5SSIV Reverse Transcriptase1 µL105.6 µL6Total7 µL739.2 µLSeal the tubes, place on ice and note the specific batch number of applied reagents in your journal
ABC1ComponentVolume pr sample96-samples2SSIV Buffer4 µL422.4 µl3100 mM DTT1 µL105.6 µL4RNaseOUT RNase Inhibitor1 µL105.6 µL5SSIV Reverse Transcriptase1 µL105.6 µL6Total7 µL739.2 µL
?. [Section 2: cDNA preparation]
Transfer the RNA samples and cDNA synthesis mastermix to the pre-PCR area.For each sample add 7 µL cDNA synthesis mastermix to the plate containing template RNA with hexamers for a total volume of 20 µL in each well.Gently mix by pipetting, seal the plate and spin down to collect liquid at the bottom of the wells.
?. [Section 2: cDNA preparation]
cDNA synthesisTransfer the plate to the PCR-machine and Incubate the reaction as follows (/protocols/COVID-19/Corona - cDNA synthesis.js):  AB1TemperatureTime242 °C50 min370 °C10 min45 °C-
AB1TemperatureTime242 °C50 min370 °C10 min45 °C-
?. [Section 2: cDNA preparation]
cDNA storageIf proceeding with the protocol the same day, temporay store the cDNA plate on ice otherwise transfer to storage at -18 °C. The plate must be labeled cDNA_CJXXX_pX if not done already.
?. [Section 3: Primer pool preparation]
Primer schemeTo effectivly amplify the entire virus genome two different primer pools with overlapping ends are needed. The primer pools used in this protocol is based on the primer sequences of the artic network version 3 (https://github.com/artic-network/artic-ncov2019/tree/master/primer_schemes/nCoV-2019/V3) with a custom scheme generating ~1200 bp amplicons with tails for easy barcoding. The primer scheme and sequences can be found under guidelines or in journals for preparing primer stocks. Pay attention to which version of the primer pool is used. For production use the newest validated version, currently V3.1.
?. [Section 3: Primer pool preparation]
Primer pool 1 and 2 (if out of stock solution)All work with stock primers must be done in the laminar flow cabinet.Primer stocks of lyophilised primers are resuspended at a concentration of 100 µM each.Primer pool stocks are generated by adding 10 µL of each primer to a 1.5 mL Epppendorf tube labelled either:    - COVID-19 long-tailed primer pool1 V3.X, 100 µM 2020-XX-XX CJXXX    - COVID-19 long-tailed primer pool2 V3.X, 100 µM 2020-XX-XX CJXXXSee guidelines/journals for preparing primer pool stocks for which primers should be included in the different pools/versions. It is recommend that multiple aliquots of each primer pool are made in case of degradation or contamination. OBS all new primer pools must be undergo QC before being applied in production.
?. [Section 3: Primer pool preparation]
Working primer solution (if out of working solution)Stock primer pools needs to be diluted 1:10 before use. This can be done just before preparing the PCR mastermix (step 14) if no more working solution is available. Dilution should be 1:10 in sterile UV treated TE buffer or NFW, to generate 10 µM primer pool working solutions.
?. [Section 4: PCR1 amplification]
PCR1 preparationsClean pre-PCR/mastermix cabinet as in step 2Consumables for PCR1Place the following items and reagents in the LAF-bench for UV-treatment (pr plate basis)2 pcs. of tear-away PCR-plates - labeled CJXXX_pX_poolX (journal, plate and pool number)24 pcs. cap-strips (wipe down plastic bag/container)1 pcs. yellow ice-block (for reagents)2 pcs 96-plate ice-block2 pcs of 1.5 mL Eppendorf tube (for primer-pool dilution)2 pcs. reservoir (for mixing of the two mastermix, one for each primer pool)Sufficient amount of each reagent depending on the amount of samples to be processed in the plate (should be calculated in template excel sheets and added to the journal)Ensure sufficient pipette tips are present (wipe new boxes down)Expose the hood to UV light for 15 minutes.UV sensitive tubes should be wiped down when entering and leaving the cabinet. Gloves should be changed each time entering and leaving the laminar flow cabinet. Production reagents must NEVER be opened outside the laminar flow cabinet.Sheet for calculating Mastermix - PCR1 amplification
?. [Section 4: PCR1 amplification]
Mastermix for targeting PCRIn the pre-PCR/master mix cabinet prepare 2 mastermix for targeting PCR as follows and alliqout to an 0.2 mL 96 well plate (tear-away) (one mastermix for each primer pool):  ABC1ComponentPr sample96 samples2Nuclease free water13.15 µL1357.08 µL35X SuperFi™ Buffer5 µL516 µL410 mM dNTP mix0.5 µL51.6 µL5Long-tailed primer pool (1 or 2) 10 µM3.6 µL371.52 µL6Platinum™ SuperFi™ DNA Polymerase (2 U/µL)0.25 µL25.8 µL7Total22.5 µL2322 µLSeal the plates with strip-caps and note the specific batch number of applied reagents in your journalSpin down to collect liquid at the bottom of the wells
ABC1ComponentPr sample96 samples2Nuclease free water13.15 µL1357.08 µL35X SuperFi™ Buffer5 µL516 µL410 mM dNTP mix0.5 µL51.6 µL5Long-tailed primer pool (1 or 2) 10 µM3.6 µL371.52 µL6Platinum™ SuperFi™ DNA Polymerase (2 U/µL)0.25 µL25.8 µL7Total22.5 µL2322 µL
?. [Section 4: PCR1 amplification]
Template additionClean cDNA area as follows: Wipe down bench, pipettes, racks, vortex, tubes, etc. in 70 % ethanol and allow to dry Wipe down bench, pipettes, racks, vortex, tubes, etc. with RNAse awayTransfer the two 96 well plates containing the mastermix for the PCR1 reaction to the cDNA work area. Spin down the plate with cDNA samples  to avoid splashing of areosls when handling.For each sample, transfer 2.5 µL cDNA sample to each well in the mastermix plate, for a final volume of 25 µL. Do so in a one column fashing to minimze risk of cross contamination:Un-seal one column of the mastermix plate and sample plateTransfer the 2.5 µL sample using a multi-pipetteRe-seal the column of the mastermix and sample plateTransfer the two 96 well plates containing the mastermix for the PCR1 reaction to the cDNA work area. Spin down the plate with cDNA samples  to avoid splashing of areosls when handling.For each sample, transfer 2.5 µL cDNA sample to each well in the mastermix plate, for a final volume of 25 µL. Do so in a one column fashing to minimze risk of cross contamination:Un-seal one column of the mastermix plate and sample plateTransfer the 2.5 µL sample using a multi-pipetteRe-seal the column of the mastermix and sample plateEach sample should be present in both pools with matching well IDs.Re-seal the cDNA plate and move to storage at -18 °C.Briefly vortex the PCR sample plates and spin down to collect liquid at the bottom of the wells.
?. [Section 4: PCR1 amplification]
PCR1 amplification conditionsSet-up the following program on two PCR machines (one for each pool), for PCR1 amplification (/protocols/COVID-19/Corona - PCR1 amplficiation.js):  ABCD1StepTemperatureTimeCycles2Heat Activation98 °C30 secsingle step3Denaturation98 °C15 sec54Annealing/Extension65 °C5 min55Hold4 °Cindefinitesingle stepSufficient tailed target amplicons are now generated for later amplification and barcoding PCR
ABCD1StepTemperatureTimeCycles2Heat Activation98 °C30 secsingle step3Denaturation98 °C15 sec54Annealing/Extension65 °C5 min55Hold4 °Cindefinitesingle step
?. [Section 5: PCR1 clean-up]
Combine pool 1 and 2Clean the barcoding area as follows: Wipe down bench, pipettes, racks, vortex, tubes, etc. in 70 % ethanol and allow to dry Wipe down bench, pipettes, racks, vortex, tubes, etc. with RNAse awayAt the barcoding area combine the contents of the PCR1 products from Pool 1 and Pool 2 for each biological sample in one of the two 96 well plates.
?. [Section 5: PCR1 clean-up]
Clean-upClean up amplicons using cleanNGS SPRI beads based on the following protocol:Step by step:Homogenize SRPI bead solution by vortexing (CleanNGS, Netherlands)Un-seal one column of samplesAdd 50 µL bead SPRI solution to each sample for a ratio of 1:1Mix by carefully pipetting up and downRepeat the three previous steps untill beads have been added to all samplesSeal the plate with foil and briefly spin down to collect liqued at the bottom of the tube (avoid pelleting of beads)Incubate at room temperature for 00:03:00Place tube on a magnetic rack and wait until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)Unseal and discard foil Carefully discard the supernatant (keep tube on the magnetic rack)Wash beads by adding 190 µL fresh Ethanol (80 % v/v) along the opposite side of the beadsWait 00:00:30 and discard the ethanol Repeat the two previous washing steps Seal the plate by applying foilSpin down plate, place it back on the magnetic rack, unseal and to remove residual ethanol Let beads air dry for no more than 00:00:30 and remove tube from the magnetic rackResuspend the purified DNA by adding 18 µL of nuclease free water.Re-seal plate with new sterile strip capsVortex the plate for mixing and lighly spin down the plate (aviod pelleting of beads)Incubate at room temperature for 00:02:00Move plate to the magnetic rack until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)
?. [Section 6: PCR2 Barcoding and amplification]
PCR2 preparationsClean pre-PCR/mastermix cabinet as in step 2Consumables for PCR2Place the following items and reagents in the LAF-bench for UV-treatment (pr plate basis)1 pcs. of tear-away PCR-plates - labeled CJXXX_pX_poolX (journal, plate and pool number)12 pcs. cap-strips (wipe down plastic bag/container)1 pcs. yellow ice-block (for reagents)1 pcs. 96-plate ice-block1 set of IDT/ILM DUAL-INDEX plate (primer with unique barcode)1 pcs. reservoir (for mixing of the of the mastermix)Sufficient amount of each reagent depending on the amount of samples to be processed in the plate (should be calculated in template excel sheets and added to the journal)Ensure sufficient pipette tips are present (wipe new boxes down)Expose the hood to UV light for 15 minutes.UV sensitive tubes should be wiped down when entering and leaving the cabinet. Gloves should be changed each time entering and leaving the laminar flow cabinet. Production reagents must NEVER be opened outside the laminar flow cabinet.Sheet for calculating Mastermix - PCR2 Barcoding and amplification
?. [Section 6: PCR2 Barcoding and amplification]
Mastermix for multiplex PCR2In the pre-PCR/master mix cabinet prepare mastermix for barcoding PCR as follows and alliqout 8 µL to each 0.2 mL well in the 96 well plate (tear-away):  ABC1ComponentPr sample96 samples2Nuclease free water2.25 µL232.2 µL35X SuperFi™ Buffer5 µL516 µL410 mM dNTP mix0.5 µL51.6 µL5ILM/IDT primer mix (10 µM)2-6Platinum™ SuperFi™ DNA Polymerase (2 U/µL)0.25 µL25.8 µL7Total10 µL825.6 µLFrom the barcode set (plate containing the ILM/IDT primer mix) transfer 2 µL of each seperate barcode primer to each seperate position in the mastermix plate, for a total volume of 10 µL. Seal the plates with strip-caps and note the specific batch number of applied reagents in your journal.Spin down to collect liquid at the bottom of the wells.Note the specific barcodes for each position of the mastermix template together with the associated sample ID in the journal.
ABC1ComponentPr sample96 samples2Nuclease free water2.25 µL232.2 µL35X SuperFi™ Buffer5 µL516 µL410 mM dNTP mix0.5 µL51.6 µL5ILM/IDT primer mix (10 µM)2-6Platinum™ SuperFi™ DNA Polymerase (2 U/µL)0.25 µL25.8 µL7Total10 µL825.6 µL
?. [Section 6: PCR2 Barcoding and amplification]
Transfer the 96 well plate containing the mastermix for the barcoding PCR2 to the barcoding work area containingthe magnetic rack with the cleaned product of PCR1.For each sample transfer 15 µL without disturbing the beads to the barcode mastermix plate for a total volume of 25 µL. Do so in a one column fashion to minimze risk of cross contamination:    - Un-seal one column of the mastermix plate and sample plate    - Transfer the 15 µL sample    - Re-seal the column of the mastermix and sample plateVortex the sealed plate to mix and spin down the plate to collect liquid at the bottom of the wells.
?. [Section 6: PCR2 Barcoding and amplification]
PCR2 conditions Set-up the following program on the PCR machine, for barcoding PCR (/protocols/COVID-19/Corona - PCR2 Barcoding Long-fragment):  ABCD1StepTemperatureTimeCycles2Heat Activation95 °C30 secsingle step3Denaturation95 °C15 sec254Annealing65 °C15 sec255Extension72 °C1 min256Final extension72 °C1 minsingle step7Hold4 °Cindefinitesingle stepThe samples are now amplified and barcoded.
ABCD1StepTemperatureTimeCycles2Heat Activation95 °C30 secsingle step3Denaturation95 °C15 sec254Annealing65 °C15 sec255Extension72 °C1 min256Final extension72 °C1 minsingle step7Hold4 °Cindefinitesingle step
?. [Section 7: PCR2 clean-up]
Preparations for clean-upClean the barcoding area as follows: Wipe down bench, pipettes, racks, vortex, tubes, etc. in 70 % ethanol and allow to dry Wipe down bench, pipettes, racks, vortex, tubes, etc. with RNAse awayAt the barcoding area clean up the barcoded amplicons using cleanNGS SPRI beads based on the following protocol:Step by step:Homogenize SRPI bead solution by vortexing (CleanNGS, Netherlands)Un-seal one column of samplesAdd 17.5 µL bead SPRI solution to each sample for a ratio of 0.7:1Mix by carefully pipetting up and downRepeat the three previous steps untill beads have been added to all samplesSeal the plate with foil and briefly spin down to collect liqued at the bottom of the tube (avoid pelleting of beads)Incubate at room temperature for 00:03:00Place tube on a magnetic rack and wait until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)Unseal and discard foil Carefully discard the supernatant (keep tube on the magnetic rack)Wash beads by adding 190 µL fresh Ethanol (80 % v/v) along the opposite side of the beadsWait 00:00:30 and discard the ethanol Repeat the two previous washing steps Seal the plate by applying foilSpin down plate, place it back on the magnetic rack, unseal and to remove residual ethanol Let beads air dry for approximately 00:00:30 and remove tube from the magnetic rackResuspend the purified DNA by adding 20 µL of nuclease free water.Re-seal plate with new sterile strip capsVortex the plate for mixing and lighly spin down the plateIncubate at room temperature for 00:02:00Move plate to the magnetic rack until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)At the barcoding area clean up the barcoded amplicons using cleanNGS SPRI beads based on the following protocol:Step by step:Homogenize SRPI bead solution by vortexing (CleanNGS, Netherlands)Un-seal one column of samplesAdd 17.5 µL bead SPRI solution to each sample for a ratio of 0.7:1Mix by carefully pipetting up and downRepeat the three previous steps untill beads have been added to all samplesSeal the plate with foil and briefly spin down to collect liqued at the bottom of the tube (avoid pelleting of beads)Incubate at room temperature for 00:03:00Place tube on a magnetic rack and wait until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)Unseal and discard foil Carefully discard the supernatant (keep tube on the magnetic rack)Wash beads by adding 190 µL fresh Ethanol (80 % v/v) along the opposite side of the beadsWait 00:00:30 and discard the ethanol Repeat the two previous washing steps Seal the plate by applying foilSpin down plate, place it back on the magnetic rack, unseal and to remove residual ethanol Let beads air dry for approximately 00:00:30 and remove tube from the magnetic rackResuspend the purified DNA by adding 20 µL of nuclease free water.Re-seal plate with new sterile strip capsVortex the plate for mixing and lighly spin down the plateIncubate at room temperature for 00:02:00Move plate to the magnetic rack until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)
?. [Section 8: Quantification, integrity and normalization]
DNA quantificationMeasure the DNA sample concentration using the TECAN instrument with the Qubit 1X dsDNA HS assay kit (Thermo Fisher Scientific) using 1 µL sample.
?. [Section 8: Quantification, integrity and normalization]
In a 96 well microtiter plate for the TECAN instrument prepare standard 1 and standard 2 by mixing:Standard 1, in well G12 (for full plates) add the following:      190 µL      Qubit™ 1X dsDNA HS Working Solution (Component A)    10 µL        Qubit™ 1X dsDNA HS Standard #1 (Component B)Standard 2, in well H12 (for full plates) add the following:    190 µL      Qubit™ 1X dsDNA HS Working Solution (Component A)    10 µL        Qubit™ 1X dsDNA HS Standard #2 (Component C)
?. [Section 8: Quantification, integrity and normalization]
For each sample mix the following in each of the remaining wells:Sample:   199 µL      Qubit™ 1X dsDNA HS Working Solution (Component A)   1 µL           Sample
?. [Section 8: Quantification, integrity and normalization]
MeasuringSeal the plate with foil and mix by vigorously vortexing for 3-5 seconds followed by a quick spin down to collect the liquid at the bottom of the wells. Allow to incubate at room temperature for 00:02:00.Carefully remove foil from the plate avoiding splashing.On the TECAN instrument press the display button to open the instrument and add the plate.On the computer open the controller software select the DNA concentration measurement script.Mark which wells to measure and assign standards (G12 and H12 for full plates). Press start.To calculate the concentrations from the measured RFU values make a standard curve from the two standards.Save the excel file in the format CJXXX_YYYY-MM-DD_PCR2 in the destination /Desktop/EB/Covid19Add concentrations to the matching sample and barcode IDs in the journal metadata sheet
?. [Section 8: Quantification, integrity and normalization]
DNA integrityMeasure the DNA integrity by gel electrophoresis on a Agilent 2200 Tapestation using D5000 screentapes (Agilent Technologies). The tape should always include the positive control and two negative control samples as awell as an ladder. Select up to 10 samples with DNA concentrations above 1.0 ng/µL for measurement. For low concentration samples the D5000 HS assay can be used to evaluate integrity.
?. [Section 8: Quantification, integrity and normalization]
Prepare the ladder in the first position of the tube-strip by mixing:     10 µL    D5000 Sample Buffer      1 µL      D5000 Ladder For each sample mix:    10 µL     D5000 Sample Buffer     1 µL       Sample
?. [Section 8: Quantification, integrity and normalization]
Vortex at 2000 rpm for 00:01:00 and spin down the tubes to collect the centents at the bottom of the tube.
?. [Section 8: Quantification, integrity and normalization]
MeasuringInsert the sample into the 2200 TapeStation Instrument.Ensure the tip-array is full with none in the trash.Select the required sample postions on the 2200 Tapestation Controller Software.Click Start and save in the format CJXXX_YYYY-MM-DD_PCR2
?. [Section 10: Normalization]
Normalization0.7 ng/µL is substracted from each sample concentration before normalization to account for dimer product. Every sample's DNA concentration is normalized to 1 ng/uL in a dilution plate by transfering 5 µL sample to an adjustable amount of nuclease free water. Diltution plates should be made with using nuclease free water with an volume calculated based on the concentratons from the metadata sheet. Skip dilution for samples with lower concentration than 1 ng/µL.
?. [Section 10: Normalization]
Pooling for sequncingFor each normalized sample transfer and pool 3 µL (3 ng of DNA). For the negative controls and possibly other sample with lower concentrations transfer and pool up to 10 µL for a maximum of 3 ng of DNA. Transfer the remaining undiluted DNA sample volumes to a skirted storage plate. Label this plate CJXXX_PCR, seal with foil and transfer to storage at -18. Concentrate the pool with a 0.7:1 SRPI bead clean-up and elute in 50 µL:Step by step:Homogenize SRPI bead solution by vortexing (CleanNGS, Netherlands)Add X µL bead SPRI solution to the pooled sample for a ratio of 0.7:1Mix by flicking the tubeBriefly spin down to collect liqued at the bottom of the tube (avoid pelleting of beads)Incubate at room temperature for 00:03:00Place tube on a magnetic rack and wait until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)Carefully discard the supernatant (keep tube on the magnetic rack)Wash beads by adding 400 µL fresh Ethanol (80 % v/v) along the opposite side of the beadsWait 00:00:30 and discard the ethanol Repeat the two previous washing stepsSpin down tube, place it back on the magnetic rack, and remove residual ethanol Let beads air dry for approximately 00:00:30 and remove tube from the magnetic rackResuspend the purified DNA by adding 50 µL of nuclease free waterVortex the tube for mixing and lighly spin down the tubeIncubate at room temperature for 00:02:00Move tube to the magnetic rack until beads have settled on the side of tube and the supernatant is completely clear (~00:00:20)
?. [Section 10: Normalization]
Library preparationThe pooled samples are prepared for MinION sequencing following the LSK-SQK 109 amplicon by ligation protocol from Nanopore using 48 µL pooled library skipping CS DNA. Keep 1 µL for integrity QC of the sample prefore and after library preparation.