- Materials
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- Input RNA in 10 mM Tris-HCl, pH 8.0
- Rapid Barcoding Kit 96 (SQK-RBK110.96)
- Midnight RT PCR Expansion (EXP-MRT001)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 5 ml Eppendorf DNA LoBind tubes
- Hard-Shell® 96-Well PCR Plates, low profile, thin-walled, skirted, white/clear (Bio-Rad, Cat # HSP9601)
- 96-well 0.8 ml MIDI plate (we recommend Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate: ThermoFisher, Cat # AB0859)
- PCR plate seals
- Hamilton 50 µl CO-RE tips with filter (Cat# 235948)
- Hamilton 300 µl CO-RE tips with filter (Cat# 235903)
- Hamilton 1000 µl CO-RE tips with filter (Cat# 235905)
- Hamilton PCR ComfortLid (Cat# 814300)
- Hamilton 60 ml Reagent Reservoir, Self-Standing with Lid (Cat# 56694-01)
- Equipment
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- Hamilton NGS STAR liquid handling robot
- Hamilton On-Deck Thermal Cycler (ODTC)
- Centrifuge capable of taking 96-well plates
- Microfuge
- Vortex mixer
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Ice bucket with ice
- Timer
- Qubit fluorometer (or equivalent)
- Optional equipment
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- Eppendorf 5424 centrifuge (or equivalent)
- PCR hood with UV steriliser (optional but recommended to reduce cross-contamination)
- PCR-Cooler (Eppendorf)
- Stepper pipette and tips
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Hamilton NGS Star 96 and deck layout
This method has been tested and validated using the Hamilton NGS Star 96 (with 8 channels and MPH96), including an on-deck thermal cycler (ODTC), a Hamilton Heater Shaker (HHS) and the Inheco Cold Plate Air Cooled (CPAC) Modules. All modules are used at different stages of the protocol. This protocol may require some fine tuning for the specific NGS Star set-up and the temperature/humidity of the customer laboratory.
Please contact your Hamilton representative for further details.
Deck layout
- MIDI plates: Abgene™ 96 Well 0.8 ml Polypropylene Deepwell Storage Plate (ThermoFisher, Cat # AB0859)
- HSP plates: Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)
- ODTC: On-Deck Thermal Cycler Module
- HHS: Hamilton Heater Shaker Module
- CPAC: Inheco Cold Plate Air Cooled Module
- 20 ml reagent troughs: X150 Reagent tub MagNA Pure LC medium 20 plastic (zigzag troughs, Roche #03004058001
- 60 ml self-standing large troughs (Hamilton, 56694-01)
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Rapid Barcoding Kit 96 (SQK-RBK110.96) contents
Name Acronym Cap colour No. of vials Fill volume per vial (µl) Rapid Barcode plate RB96 - 3 plates 8 µl per well AMPure XP Beads AXP Brown 3 1,200 Sequencing Buffer II SBII Red 1 500 Rapid Adapter F RAP-F Green 1 25 Elution Buffer EB Black 1 500 Loading Beads II LBII Pink 1 360 Loading Solution LS White cap, pink label 1 400 Flush Tether FLT Purple 1 400 Flush Buffer FB White 1 bottle 15,500 This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc.
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Midnight RT PCR Expansion (EXP-MRT001) contents
Name Acronym Cap colour Number of vials Fill volume per vial (µl) LunaScript RT SuperMix LS RT Blue 3 500 Q5 HS Master Mix Q5 Orange 6 1,500 Midnight Primer Pool A MP A White 3 15 Midnight Primer Pool B MP B Clear 3 15 -
Midnight Primer sequences
As mutations in SARS-CoV-2 variants emerge amplicon drop out may be observed; for users wishing to design their own primer spike-ins to address this we suggest adding to the appropriate primer pool at a final concentration between 3.33 µM and 6.66 µM.
Below are the sequences for the V3 primer scheme used in the Midnight RT PCR Expansion.
Pool A
Primer name Primer Sequence SARSCoV_1200_1_LEFT
ACCAACCAACTTTCGATCTCTTGT SARSCoV_1200_1_RIGHT
GGTTGCATTCATTTGGTGACGC SARSCoV_1200_3_LEFT
GGCTTGAAGAGAAGTTTAAGGAAGGT SARSCoV_1200_3_RIGHT
GATTGTCCTCACTGCCGTCTTG SARSCoV_1200_5_LEFT
ACCTACTAAAAAGGCTGGTGGC SARSCoV_1200_5_RIGHT
AGCATCTTGTAGAGCAGGTGGA SARSCoV_1200_7_LEFT
ACCTGGTGTATACGTTGTCTTTGG SARSCoV_1200_7_RIGHT
GCTGAAATCGGGGCCATTTGTA SARSCoV_1200_9_LEFT
AGAAGTTACTGGCGATAGTTGTAATAACT SARSCoV_1200_9_RIGHT
TGCTGATATGTCCAAAGCACCA SARSCoV_1200_11_LEFT
AGACACCTAAGTATAAGTTTGTTCGCA SARSCoV_1200_11_RIGHT
GCCCACATGGAAATGGCTTGAT SARSCoV_1200_13_LEFT
ACCTCTTACAACAGCAGCCAAAC SARSCoV_1200_13_RIGHT
CGTCCTTTTCTTGGAAGCGACA SARSCoV_1200_15_LEFT
TTTTAAGGAATTACTTGTGTATGCTGCT SARSCoV_1200_15_RIGHT
ACACACAACAGCATCGTCAGAG SARSCoV_1200_17_LEFT
TCAAGCTTTTTGCAGCAGAAACG SARSCoV_1200_17_RIGHT
CCAAGCAGGGTTACGTGTAAGG SARSCoV_1200_19_LEFT
GGCACATGGCTTTGAGTTGACA SARSCoV_1200_19_RIGHT
CCTGTTGTCCATCAAAGTGTCCC SARSCoV_1200_21_LEFT
TCTGTAGTTTCTAAGGTTGTCAAAGTGA SARSCoV_1200_21_RIGHT
GCAGGGGGTAATTGAGTTCTGG 21_right_spike
GTGTATGATTGAGTTCTGGTTGTAAG SARSCoV_1200_23_LEFT
ACTTTAGAGTCCAACCAACAGAATCT 23_left_spike
ACTTTAGAGTTCAACCAACAGAATCT SARSCoV_1200_23_RIGHT
TGACTAGCTACACTACGTGCCC SARSCoV_1200_25_LEFT
TGCTGCTACTAAAATGTCAGAGTGT SARSCoV_1200_25_RIGHT
CATTTCCAGCAAAGCCAAAGCC SARSCoV_1200_27_LEFT
TGGATCACCGGTGGAATTGCTA SARSCoV_1200_27_RIGHT
TGTTCGTTTAGGCGTGACAAGT SARSCoV_1200_29_LEFT
TGAGGGAGCCTTGAATACACCA SARSCoV_1200_29_RIGHT
TAGGCAGCTCTCCCTAGCATTG Pool B
Primer name Primer sequences SARSCoV_1200_2_LEFT
CCATAATCAAGACTATTCAACCAAGGGT SARSCoV_1200_2_RIGHT
ACAGGTGACAATTTGTCCACCG SARSCoV_1200_4_LEFT
GGAATTTGGTGCCACTTCTGCT SARSCoV_1200_4_RIGHT
CCTGACCCGGGTAAGTGGTTAT SARSCoV_1200_6_LEFT
ACTTCTATTAAATGGGCAGATAACAACTG SARSCoV_1200_6_RIGHT
GATTATCCATTCCCTGCGCGTC SARSCoV_1200_8_LEFT
CAATCATGCAATTGTTTTTCAGCTATTTTG SARSCoV_1200_8_RIGHT
TGACTTTTTGCTACCTGCGCAT SARSCoV_1200_10_LEFT
TTTACCAGGAGTTTTCTGTGGTGT SARSCoV_1200_10_RIGHT
TGGGCCTCATAGCACATTGGTA SARSCoV_1200_12_LEFT
ATGGTGCTAGGAGAGTGTGGAC SARSCoV_1200_12_RIGHT
GGATTTCCCACAATGCTGATGC SARSCoV_1200_14_LEFT
ACAGGCACTAGTACTGATGTCGT SARSCoV_1200_14_RIGHT
GTGCAGCTACTGAAAAGCACGT SARSCoV_1200_16_LEFT
ACAACACAGACTTTATGAGTGTCTCT SARSCoV_1200_16_RIGHT
CTCTGTCAGACAGCACTTCACG SARSCoV_1200_18_LEFT
GCACATAAAGACAAATCAGCTCAATGC SARSCoV_1200_18_RIGHT
TGTCTGAAGCAGTGGAAAAGCA SARSCoV_1200_20_LEFT
ACAATTTGATACTTATAACCTCTGGAACAC SARSCoV_1200_20_RIGHT
GATTAGGCATAGCAACACCCGG SARSCoV_1200_22_LEFT
GTGATGTTCTTGTTAACAACTAAACGAACA SARSCoV_1200_22_RIGHT
AACAGATGCAAATCTGGTGGCG 22_right_spike
AACAGATGCAAATTTGGTGGCG SARSCoV_1200_24_LEFT
GCTGAACATGTCAACAACTCATATGA 24_left_spike
GCTGAATATGTCAACAACTCATATGA SARSCoV_1200_24_RIGHT
ATGAGGTGCTGACTGAGGGAAG SARSCoV_1200_26_LEFT
GCCTTGAAGCCCCTTTTCTCTA SARSCoV_1200_26_RIGHT
AATGACCACATGGAACGCGTAC SARSCoV_1200_28_LEFT
TTTGTGCTTTTTAGCCTTTCTGCT SARSCoV_1200_28_RIGHT
GTTTGGCCTTGTTGTTGTTGGC SARSCoV_1200_28_LEFT_27837T
TTTGTGCTTTTTAGCCTTTCTGTT -
Rapid barcode sequences
Component Sequence RB01 AAGAAAGTTGTCGGTGTCTTTGTG RB02 TCGATTCCGTTTGTAGTCGTCTGT RB03 GAGTCTTGTGTCCCAGTTACCAGG RB04 TTCGGATTCTATCGTGTTTCCCTA RB05 CTTGTCCAGGGTTTGTGTAACCTT RB06 TTCTCGCAAAGGCAGAAAGTAGTC RB07 GTGTTACCGTGGGAATGAATCCTT RB08 TTCAGGGAACAAACCAAGTTACGT RB09 AACTAGGCACAGCGAGTCTTGGTT RB10 AAGCGTTGAAACCTTTGTCCTCTC RB11 GTTTCATCTATCGGAGGGAATGGA RB12 CAGGTAGAAAGAAGCAGAATCGGA RB13 AGAACGACTTCCATACTCGTGTGA RB14 AACGAGTCTCTTGGGACCCATAGA RB15 AGGTCTACCTCGCTAACACCACTG RB16 CGTCAACTGACAGTGGTTCGTACT RB17 ACCCTCCAGGAAAGTACCTCTGAT RB18 CCAAACCCAACAACCTAGATAGGC RB19 GTTCCTCGTGCAGTGTCAAGAGAT RB20 TTGCGTCCTGTTACGAGAACTCAT RB21 GAGCCTCTCATTGTCCGTTCTCTA RB22 ACCACTGCCATGTATCAAAGTACG RB23 CTTACTACCCAGTGAACCTCCTCG RB24 GCATAGTTCTGCATGATGGGTTAG RB25 GTAAGTTGGGTATGCAACGCAATG RB26 CATACAGCGACTACGCATTCTCAT RB27 CGACGGTTAGATTCACCTCTTACA RB28 TGAAACCTAAGAAGGCACCGTATC RB29 CTAGACACCTTGGGTTGACAGACC RB30 TCAGTGAGGATCTACTTCGACCCA RB31 TGCGTACAGCAATCAGTTACATTG RB32 CCAGTAGAAGTCCGACAACGTCAT RB33 CAGACTTGGTACGGTTGGGTAACT RB34 GGACGAAGAACTCAAGTCAAAGGC RB35 CTACTTACGAAGCTGAGGGACTGC RB36 ATGTCCCAGTTAGAGGAGGAAACA RB37 GCTTGCGATTGATGCTTAGTATCA RB38 ACCACAGGAGGACGATACAGAGAA RB39 CCACAGTGTCAACTAGAGCCTCTC RB40 TAGTTTGGATGACCAAGGATAGCC RB41 GGAGTTCGTCCAGAGAAGTACACG RB42 CTACGTGTAAGGCATACCTGCCAG RB43 CTTTCGTTGTTGACTCGACGGTAG RB44 AGTAGAAAGGGTTCCTTCCCACTC RB45 GATCCAACAGAGATGCCTTCAGTG RB46 GCTGTGTTCCACTTCATTCTCCTG RB47 GTGCAACTTTCCCACAGGTAGTTC RB48 CATCTGGAACGTGGTACACCTGTA RB49 ACTGGTGCAGCTTTGAACATCTAG RB50 ATGGACTTTGGTAACTTCCTGCGT RB51 GTTGAATGAGCCTACTGGGTCCTC RB52 TGAGAGACAAGATTGTTCGTGGAC RB53 AGATTCAGACCGTCTCATGCAAAG RB54 CAAGAGCTTTGACTAAGGAGCATG RB55 TGGAAGATGAGACCCTGATCTACG RB56 TCACTACTCAACAGGTGGCATGAA RB57 GCTAGGTCAATCTCCTTCGGAAGT RB58 CAGGTTACTCCTCCGTGAGTCTGA RB59 TCAATCAAGAAGGGAAAGCAAGGT RB60 CATGTTCAACCAAGGCTTCTATGG RB61 AGAGGGTACTATGTGCCTCAGCAC RB62 CACCCACACTTACTTCAGGACGTA RB63 TTCTGAAGTTCCTGGGTCTTGAAC RB64 GACAGACACCGTTCATCGACTTTC RB65 TTCTCAGTCTTCCTCCAGACAAGG RB66 CCGATCCTTGTGGCTTCTAACTTC RB67 GTTTGTCATACTCGTGTGCTCACC RB68 GAATCTAAGCAAACACGAAGGTGG RB69 TACAGTCCGAGCCTCATGTGATCT RB70 ACCGAGATCCTACGAATGGAGTGT RB71 CCTGGGAGCATCAGGTAGTAACAG RB72 TAGCTGACTGTCTTCCATACCGAC RB73 AAGAAACAGGATGACAGAACCCTC RB74 TACAAGCATCCCAACACTTCCACT RB75 GACCATTGTGATGAACCCTGTTGT RB76 ATGCTTGTTACATCAACCCTGGAC RB77 CGACCTGTTTCTCAGGGATACAAC RB78 AACAACCGAACCTTTGAATCAGAA RB79 TCTCGGAGATAGTTCTCACTGCTG RB80 CGGATGAACATAGGATAGCGATTC RB81 CCTCATCTTGTGAAGTTGTTTCGG RB82 ACGGTATGTCGAGTTCCAGGACTA RB83 TGGCTTGATCTAGGTAAGGTCGAA RB84 GTAGTGGACCTAGAACCTGTGCCA RB85 AACGGAGGAGTTAGTTGGATGATC RB86 AGGTGATCCCAACAAGCGTAAGTA RB87 TACATGCTCCTGTTGTTAGGGAGG RB88 TCTTCTACTACCGATCCGAAGCAG RB89 ACAGCATCAATGTTTGGCTAGTTG RB90 GATGTAGAGGGTACGGTTTGAGGC RB91 GGCTCCATAGGAACTCACGCTACT RB92 TTGTGAGTGGAAAGATACAGGACC RB93 AGTTTCCATCACTTCAGACTTGGG RB94 GATTGTCCTCAAACTGCCACCTAC RB95 CCTGTCTGGAAGAAGAATGGACTT RB96 CTGAACGGTCATAGAGTCCACCAT