TITLE: Ebola virus sequencing protocol
AUTHORS: Josh Quick

[STEPS]
?. [cDNA preparation]
Mix the following components in an 0.2mL 8-strip tube;Component                                        Volume50µM random hexamers                 10mM dNTPs mix (10mM each)    Template RNA                                   Total
1 µl
1 µl
10 µl
12 µl
Viral RNA input from a clinical sample should be between Ct 18-35. If Ct is between 12-15, then dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows: for Place on ice for
65 °C
?. [cDNA preparation]
Add the following to the annealed template RNA: Component	                                Volume SSIV Buffer	                                 100mM DTT                                  RNaseOUT RNase Inhibitor 	 SSIV Reverse Transcriptase	 Total
4 µl
1 µl
1 µl
1 µl
20 µl
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows:Hold at
42 °C
70 °C
5 °C
?. [Primer pool preparation]
If required resuspend lyophilised primers at a concentration of 100µM each
Ebola V2 primers for this protocol were designed using Primal Scheme and generate overlapping 400nt amplicons. Primer names and dilutions are listed in the table below.
?. [Primer pool preparation]
Generate primer pool stocks by adding  of each primer pair to a  Eppendorf labelled either “Pool 1 (100µM)” or “Pool 2 (100µM)”. Total volume should be  for Pool 1 (100µM) and  for Pool 2 (100µM). These are your 100µM stocks of each primer pool.
5 µl
1.5 ml
505 µl
530 µl
Primers should be prepped and aliquoted prior to departure in a sterile PCR cabinet. At no stage should primers or PCR reagents be anywhere near the template until cDNA addition.
?. [Primer pool preparation]
Dilute this primer pool 1:10 in molecular grade water, to generate 10µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.  ABCDEF1NameSequenceNameSequencePoolStock2Ebov-10-Pan_1_LEFTTGTGTGCGAATAACTATGAGGAAGAEbov-10-Pan_1_RIGHTTTTCCAATGTTTTACCCCAAGCTTT1100µM3Ebov-10-Pan_1_RIGHT_alt1TTTCCAATGCTTTACCCCAAGCTTT1100µM4Ebov-10-Pan_1_RIGHT_alt2TTTCCAATGTTTTACCCCAAGTTTT1100µM5Ebov-10-Pan_2_LEFTCAAGCAAGATTGAGAATTAACCTTGGTEbov-10-Pan_2_RIGHTATCTCCCTGGTACGCATGATGA2100µM6Ebov-10-Pan_2_LEFT_alt1CAAGCAAGATTGAGAATTAACCTTGATEbov-10-Pan_2_RIGHT_alt1ATCTCCTTGGTACGCATGATGA2100µM7Ebov-10-Pan_3_LEFTGGCCTTTGAAGCAGGTGTTGATEbov-10-Pan_3_RIGHTTCAGTCCTTGCTCTGCATGTAC1100µM8Ebov-10-Pan_4_LEFTCCTTTGCAAGTCTATTCCTTCCGAEbov-10-Pan_4_RIGHTCTGAGTGCAGCCTTAAAGGAGT2100µM9Ebov-10-Pan_4_LEFT_alt1CTTTTGCAAGTCTATTCCTTCCGA2100µM10Ebov-10-Pan_5_LEFTAGTTCGTCTCCATCCTCTTGCAEbov-10-Pan_5_RIGHTCTGGAAGCTGATTTCGTTCTTTTTCT1100µM11Ebov-10-Pan_6_LEFTGAGTCTCGCGAACTTGACCATCEbov-10-Pan_6_RIGHTTCCTCGTCGTCCTCGTCTAGAT2100µM12Ebov-10-Pan_6_LEFT_alt1GAATCTCGCGAACTTGACCATCEbov-10-Pan_6_RIGHT_alt1TCCTCATCGTCCTCGTCTAGAT2100µM13Ebov-10-Pan_7_LEFTAGCTACGGCGAATACCAGAGTTEbov-10-Pan_7_RIGHTGTCCCTGTCCTGCTCTTCATCA1100µM14Ebov-10-Pan_7_RIGHT_alt1GTCCCTGTCCTGTTCTTCATCA1100µM15Ebov-10-Pan_7_RIGHT_alt2GTCCCTGTCCTGTTCTTCATCG1100µM16Ebov-10-Pan_8_LEFTTTAACGAAGAGGCAGACCCACTEbov-10-Pan_8_RIGHTTTCCTCTTCAAGGGAGTCTGGA2100µM17Ebov-10-Pan_8_LEFT_alt1TCAACGAAGAGGCAGACCCACTEbov-10-Pan_8_RIGHT_alt1TTCCTCTTCAAGGGAGTCCGGA2100µM18Ebov-10-Pan_9_LEFTGTGACAACACCCAGTCAGAACAEbov-10-Pan_9_RIGHTTCTTCCTGTTTTCGTTCCTTGACT1100µM19Ebov-10-Pan_9_LEFT_alt1GTGACAACACCCAGCCAGAACAEbov-10-Pan_9_RIGHT_alt1TCTTCCTGTTTGCGTTCCTTGACT1100µM20Ebov-10-Pan_9_RIGHT_alt2TCTTCCTGTTTGCGTTTCTTGACT1100µM21Ebov-10-Pan_10_LEFTACAATGGGATGATTCAACCGACAEbov-10-Pan_10_RIGHTTCGAGTGCTAGAGAATTCAATTGACG2100µM22Ebov-10-Pan_10_LEFT_alt1ATAATGGGATGATTTAACCGACA2100µM23Ebov-10-Pan_11_LEFTACCTACTAGCCTGCCCAACATTEbov-10-Pan_11_RIGHTAATTGGGTCCGTTTGGGTTTGA1100µM24Ebov-10-Pan_11_LEFT_alt1ACCTACTAGCCTACCCAACATTEbov-10-Pan_11_RIGHT_alt1AATTGGATCCGTTTGGGTTTGA1100µM25Ebov-10-Pan_12_LEFTCCCAAATGCAACAAACGAAGCCEbov-10-Pan_12_RIGHTTCAATCTTACCCCGAATCGCAC2100µM26Ebov-10-Pan_12_LEFT_alt1CCCAAATGCAACAAACAAAGCCEbov-10-Pan_12_RIGHT_alt1TCAATCTTACCCCGAATTGCAC2100µM27Ebov-10-Pan_13_LEFTTATTGGGCCGAACATGGTCAACEbov-10-Pan_13_RIGHTTGACAGGTGGAGCAGCATCTTG1100µM28Ebov-10-Pan_13_LEFT_alt1TATTGGGCTGAACATGGTCAAC1100µM29Ebov-10-Pan_14_LEFTCATTCATGCTGAGTTCCAGGCCEbov-10-Pan_14_RIGHTGCGAGATATGAACAATTTTATCTTGGTCG2100µM30Ebov-10-Pan_14_RIGHT_alt1GCGAGATAAGGACAATTTTATCTTGGTCG2100µM31Ebov-10-Pan_14_RIGHT_alt2GCGAGATAAGAACAATTTTATCTTGGTCG2100µM32Ebov-10-Pan_15_LEFTTGAGTATCAGCCCTGGATAATATAAGTCAEbov-10-Pan_15_RIGHTTCGATGGAGTGTCCCCATTGAC1100µM33Ebov-10-Pan_15_LEFT_alt1TGAGTATCAGCCCTAGATAATATAAGTCAEbov-10-Pan_15_RIGHT_alt1TCGATGGAGTGTCTCCATTGAC1100µM34Ebov-10-Pan_16_LEFTGCAACAGCAATACAGGCTTCCTEbov-10-Pan_16_RIGHTGAAAGCCTGGTTTCCAATTCGC2100µM35Ebov-10-Pan_16_LEFT_alt1GCAACAACAATACAGGCTTCCTEbov-10-Pan_16_RIGHT_alt1GAAGGCCTGGTTTCCAATTCGC2100µM36Ebov-10-Pan_17_LEFTCCACTTGTCAGAGTCAATCGGCEbov-10-Pan_17_RIGHTGTTTCTGGCACTTCGATTCCCA1100µM37Ebov-10-Pan_17_RIGHT_alt1GTTTCTGGCACTTCGATACCCA1100µM38Ebov-10-Pan_18_LEFTAAAATCCAAGCAATAATGACTTCACTCCEbov-10-Pan_18_RIGHTTTGATCAATTAAAAGTGTCTCCTCTAATGG2100µM39Ebov-10-Pan_18_RIGHT_alt1TCGATCAATTTAAAGTATCTCCTCTAATGG2100µM40Ebov-10-Pan_18_RIGHT_alt2TTGATCAATTAAAAGTATCTCCTCTAATAG2100µM41Ebov-10-Pan_19_LEFTAGATCCAGTTTTATAGAATCTTCTCAGGGAEbov-10-Pan_19_RIGHTAGAAGGGCAATGTCTGTACTTGG1100µM42Ebov-10-Pan_19_LEFT_alt1AGATCCAGTTTTACAGAATCTTCTCAGGGAEbov-10-Pan_19_RIGHT_alt1AGAAGGGCGATGTCTGTGCTTGG1100µM43Ebov-10-Pan_20_LEFTAGCCAGTGTGACTTGGATTGGAEbov-10-Pan_20_RIGHTAGTTTGTCGACATCACTAACCTGT2100µM44Ebov-10-Pan_20_RIGHT_alt1AGTTTGTCGACATCACTAACTTGT2100µM45Ebov-10-Pan_21_LEFTAGAACATTTTCCATCCCACTTGGAEbov-10-Pan_21_RIGHTAAGCACCCTCTTTATGGAAGGC1100µM46Ebov-10-Pan_21_RIGHT_alt1AAGCACCCTCTTTGTGGAAGGC1100µM47Ebov-10-Pan_22_LEFTTGCCGGTATGTGCACAAAGTATEbov-10-Pan_22_RIGHTATATATTGTCTCATTCAGCTGGAGCA2100µM48Ebov-10-Pan_23_LEFTCGAGGTTGACAATTTGACCTACGTEbov-10-Pan_23_RIGHTGCAAGGGTTGTTAGATGCGACA1100µM49Ebov-10-Pan_23_RIGHT_alt1GCAAGGGTTGTCAGATGCGACA1100µM50Ebov-10-Pan_24_LEFTTGCAATGGTTCAAGTGCACAGTEbov-10-Pan_24_RIGHTCTGGCACTCTCTTCTCCGGTAT2100µM51Ebov-10-Pan_24_LEFT_alt1TGCAATGGTTCAAGTGCACAAT2100µM52Ebov-10-Pan_25_LEFTACCACAACAAGTCCCCAAAACCEbov-10-Pan_25_RIGHTTAGCTCAGTTGTGGCTCTCAGG1100µM53Ebov-10-Pan_25_RIGHT_alt1TAGCTCGGTTGTGGCTCTCAGG1100µM54Ebov-10-Pan_26_LEFTATCTGTGGGTTGAGACAGCTGGEbov-10-Pan_26_RIGHTGCTTTTCCATGAAGCAATCTGAAGA2100µM55Ebov-10-Pan_26_LEFT_alt1ATCTGTGGATTGAGGCAGCTGGEbov-10-Pan_26_RIGHT_alt1GCTTTGCCATGAAGCAATCTGAAGA2100µM56Ebov-10-Pan_26_LEFT_alt2ATCTGTGGGTTGAGGCAGCTGG2100µM57Ebov-10-Pan_27_LEFTTGGAGTTACAGGCGTTATAATTGCAEbov-10-Pan_27_RIGHTAAAGGCTTCTTTCCCTTGTCACT1100µM58Ebov-10-Pan_28_LEFTTCATCCTTGATTCTACAATCATGACAGTEbov-10-Pan_28_RIGHTAGGTGCTGGAGGAACTGTTAATG2100µM59Ebov-10-Pan_28_LEFT_alt1TCATCCTTGATTCTACAATCATAACAGT2100µM60Ebov-10-Pan_29_LEFTGAGTACCGTCAATCAAGGAGCGEbov-10-Pan_29_RIGHTCACAGCACATAGAGTCAACAATGC1100µM61Ebov-10-Pan_30_LEFTGATCAAGACGGCAGAACACTGGEbov-10-Pan_30_RIGHTATCAGACCATGAGCATGTCCCC2100µM62Ebov-10-Pan_31_LEFTCTGCTGTCGTTGTTTCAGGGTTEbov-10-Pan_31_RIGHTATGGGATGGATCGTTGCTACCT1100µM63Ebov-10-Pan_31_RIGHT_alt1ATGGGATGGATCGTTGCTGCCT1100µM64Ebov-10-Pan_31_RIGHT_alt2ATGAGATGGATCGTTGCTACCT1100µM65Ebov-10-Pan_32_LEFTGCCAAGCATACCTCTTGCACAAEbov-10-Pan_32_RIGHTTGGACTACCCTGAAATAGTACTTTGC2100µM66Ebov-10-Pan_33_LEFTTGCGGAGGTCTGATAAGAATAAACCEbov-10-Pan_33_RIGHTTTCAACCTTGAAACCTTGCGCT1100µM67Ebov-10-Pan_33_RIGHT_alt1TTCAACTTTGAAACCTTGCGCT1100µM68Ebov-10-Pan_34_LEFTGCTGAAAAGAAGCTTACCTACAACGEbov-10-Pan_34_RIGHTTCCTTGTCATTGACCATGCAGG2100µM69Ebov-10-Pan_34_LEFT_alt1GTTGAAAAAAGGCCTACCTACAACG2100µM70Ebov-10-Pan_34_LEFT_alt2GCTGAAAAGAAGCCCACCTACAACG2100µM71Ebov-10-Pan_35_LEFTGTGACTCACAAAGGAATGGCCCEbov-10-Pan_35_RIGHTACAATCCGTTGTAGTTCACGACA1100µM72Ebov-10-Pan_35_RIGHT_alt1ACAACCCGTTGTAGTTCACGACA1100µM73Ebov-10-Pan_36_LEFTTGCTGTCGTTGATTCGATCCAAEbov-10-Pan_36_RIGHTAGCAGAGATGTCAAGATAACTATTGAGT2100µM74Ebov-10-Pan_37_LEFTACACGAATGCAAAGTTTGATTCTTGAEbov-10-Pan_37_RIGHTTGAAACCTAACACATGTGACCTGC1100µM75Ebov-10-Pan_37_RIGHT_alt1TGAAACCTAACACACGTGACCTGC1100µM76Ebov-10-Pan_38_LEFTCCCTCAAACAAGAGATTCCAAGACAEbov-10-Pan_38_RIGHTACAGTTGCGTAGTTGCGGATTA2100µM77Ebov-10-Pan_38_LEFT_alt1CCCTCAAATAAGAGATTCCAAGACA2100µM78Ebov-10-Pan_38_LEFT_alt2TCCTCAAATAAGAGATTCCAAGACA2100µM79Ebov-10-Pan_39_LEFTACCTAGTCACTAGAGCTTGCGGEbov-10-Pan_39_RIGHTACATTTGATGTAAAAATTCATTGCCCTG1100µM80Ebov-10-Pan_40_LEFTGTGGGTGCTCAAGAAGACTGTGEbov-10-Pan_40_RIGHTTGAGATTAGAGTTGTGTTGAATCGACA2100µM81Ebov-10-Pan_40_LEFT_alt1GTGGGTGCTCAAGAGGACTGTGEbov-10-Pan_40_RIGHT_alt1TGAGATTAGAGTCGTGTTGAATCGACA2100µM82Ebov-10-Pan_41_LEFTAAGAAGCGGTTCAAGGGCATACEbov-10-Pan_41_RIGHTCTATGGAATTCACGGATCTTTTGAGC1100µM83Ebov-10-Pan_41_LEFT_alt1AAGAAGCAGTTCAAGGGCATACEbov-10-Pan_41_RIGHT_alt1CTATGGAATTCACGGATCTTTTGATC1100µM84Ebov-10-Pan_42_LEFTTGCATTTAGCTGTAAATCACACCCTEbov-10-Pan_42_RIGHTAATCATTGGCAACGGAGGGAAT2100µM85Ebov-10-Pan_42_RIGHT_alt1AATCATTGGCAACGGGGGGAAT2100µM86Ebov-10-Pan_43_LEFTGTCAAGGATCTTGGTACAGTGTTACTEbov-10-Pan_43_RIGHTTGAGAAAGAAAAGTTCCGATATTGTGGT1100µM87Ebov-10-Pan_43_LEFT_alt1GCCAAGGGTCTTGGTACAGTGTTACTEbov-10-Pan_43_RIGHT_alt1TGAGAAAGAAAAATTCCGGTATTGTGGT1100µM88Ebov-10-Pan_43_LEFT_alt2GTCAAGGGTCTTGGTACAGTGTTACTEbov-10-Pan_43_RIGHT_alt2TGAGAAAGAAAAATTCCGATATTGTGGT1100µM89Ebov-10-Pan_44_LEFTTTGAGAATGTTCTTTCCTACGCACAEbov-10-Pan_44_RIGHTACGGTTGCAATATTCTATAAAAGGTGC2100µM90Ebov-10-Pan_44_LEFT_alt1TTGAGAATGTTCTTTCCTACGCGCAEbov-10-Pan_44_RIGHT_alt1ACGGTTGCAATATTCGATAAAAGGTGC2100µM91Ebov-10-Pan_44_RIGHT_alt2ACGGTTACAATATTCTATAAAAGGTGC2100µM92Ebov-10-Pan_45_LEFTCCACAGTTAGAGGGAGTAGCTTTGEbov-10-Pan_45_RIGHTGCTCGTCTGCGTCAGTCTCTAA1100µM93Ebov-10-Pan_45_LEFT_alt1CCACAGTTAGAGGGAGTAGTTTTG1100µM94Ebov-10-Pan_46_LEFTAAGTTACGCTCAGCTGTGATGGEbov-10-Pan_46_RIGHTATGGAAAGCTGCGGTTATCCTG2100µM95Ebov-10-Pan_47_LEFTTAGGCACTGCTTTTGAGCGATCEbov-10-Pan_47_RIGHTCACAAAGTCAATGGCAGTGCAG1100µM96Ebov-10-Pan_47_LEFT_alt1TAGGCACCGCTTTTGAGCGGTC1100µM97Ebov-10-Pan_47_LEFT_alt2TAGGCACTGCTTTTGAACGATC1100µM98Ebov-10-Pan_48_LEFTTCTCCGAATGATTGAGATGGATGATTEbov-10-Pan_48_RIGHTCTCAGTCTGTCCAAAACCGGTG2100µM99Ebov-10-Pan_48_LEFT_alt1TCTCCGAATGATTGGGATGGATGATT2100µM100Ebov-10-Pan_49_LEFTGATATCTTTTCACGCACGCCGAEbov-10-Pan_49_RIGHTCCACCTGGTTGCTTTGCATTTG1100µM101Ebov-10-Pan_49_LEFT_alt1GATATCTTTTCACGCACGCCCAEbov-10-Pan_49_RIGHT_alt1CCACCAGGTTGCTTTGCATTTG1100µM102Ebov-10-Pan_50_LEFTTCAAAGTGTTTTGGCTGAAACCCTEbov-10-Pan_50_RIGHTTCCTGAGTAATGTGAAGGGGTCA2100µM103Ebov-10-Pan_50_LEFT_alt1TCAAAGTGGTTTGGCTGAAACCCTEbov-10-Pan_50_RIGHT_alt1TCCTGAGTAATGTGAAGGAGTCA2100µM104Ebov-10-Pan_51_LEFTAACAGTGACTTGCTAATAAAACCATTTTTGEbov-10-Pan_51_RIGHTAAATACTGAGCTGGTACTTCCCG1100µM105Ebov-10-Pan_51_LEFT_alt1AACAGTGACTTGCTAATAAAGCCATTTTTG1100µM106Ebov-10-Pan_51_LEFT_alt2AACAGTGATTTGCTAATAAAACCATTTTTG1100µM107Ebov-10-Pan_52_LEFTAATCGTGCTCACCTTCATCTAACTEbov-10-Pan_52_RIGHTCCCAAAACTGTACAGAAGTCCTATCT2100µM108Ebov-10-Pan_53_LEFTACAGACCCAATTAGCAGTGGAGAEbov-10-Pan_53_RIGHTACAATTGTTCCGCGATTAATTATCCAT1100µM109Ebov-10-Pan_53_LEFT_alt1ACAGACCCAATTAGCAGCGGAGAEbov-10-Pan_53_RIGHT_alt1ACAATTGTTCCGCGATTAATTATCCACT1100µM110Ebov-10-Pan_54_LEFTTCTCAGATGCGGCCAGGTTATTEbov-10-Pan_54_RIGHTTGACCATCACTGTTGTTTGTGCT2100µM111Ebov-10-Pan_54_LEFT_alt1TCTCAGATGCGGCCAGATTATT2100µM112Ebov-10-Pan_55_LEFTTGGAGGAGCAGACACAGAAACAEbov-10-Pan_55_RIGHTATGACGTTAATTGGCGTGTCCC1100µM113Ebov-10-Pan_55_LEFT_alt1TGGAGGAGCAGGCACAGAAACAEbov-10-Pan_55_RIGHT_alt1ATGACGTCAATTGGCGTGTCCC1100µM114Ebov-10-Pan_55_LEFT_alt2TGGAGAAGCAGGCACAGAAACAEbov-10-Pan_55_RIGHT_alt2ATGACGTTAATTGGCGCGTCCC1100µM115Ebov-10-Pan_56_LEFTCTCACACCGTCTAGTCCTACCTEbov-10-Pan_56_RIGHTTTTGACATAACAGGTAGAAGCATCCT2100µM116Ebov-10-Pan_56_LEFT_alt1CTCGCACCGTCTAGTCCTACCT2100µM117Ebov-10-Pan_56_LEFT_alt2CTCACATCGTCTAGTCCTACCT2100µM118Ebov-10-Pan_57_LEFTACACGCTAGCTACTGAGTCCAGEbov-10-Pan_57_RIGHTATTGGCTTAATTAAATAACCAGTGGCA1100µM119Ebov-10-Pan_58_LEFTTGAAAGCAGTGGTCCTTAAAGTCTEbov-10-Pan_58_RIGHTTGCTCTAAGATGTGCTAAGTGCTG2100µM120Ebov-10-Pan_58_RIGHT_alt1TGCTCTAAGATGTGCCAAGTGCTG2100µM121Ebov-10-Pan_59_LEFTCGTCGATTCAAAAAGAGGTCCACTEbov-10-Pan_59_RIGHTTCAGAAGCCCTGTCAGCCTTTC1100µM122Ebov-10-Pan_60_LEFTAGATTGCAATTGTGAAGAACGTTTCTEbov-10-Pan_60_RIGHTAGAGTGCAGAGTTTATTATGTTGCGT2100µM123Ebov-10-Pan_61_LEFTTCACAATGCAGCATGTGTGACAEbov-10-Pan_61_RIGHTAGGTATTTCTGATTTTACAGTCCTGCC1100µM124Ebov-10-Pan_61_RIGHT_alt1AGGTATTTATGATTTTACAGTCCTGCC1100µM125Ebov-10-Pan_61_RIGHT_alt2AGGTATTTCTGATTTTACAGTCATGCC1100µM126Ebov-10-Pan_62_LEFTCCTGTCAGATGGAATAGTGTTTTGGTEbov-10-Pan_62_RIGHTAATTTTTGTGTGCGACCATTTTTCC2100µM
ABCDEF1NameSequenceNameSequencePoolStock2Ebov-10-Pan_1_LEFTTGTGTGCGAATAACTATGAGGAAGAEbov-10-Pan_1_RIGHTTTTCCAATGTTTTACCCCAAGCTTT1100µM3Ebov-10-Pan_1_RIGHT_alt1TTTCCAATGCTTTACCCCAAGCTTT1100µM4Ebov-10-Pan_1_RIGHT_alt2TTTCCAATGTTTTACCCCAAGTTTT1100µM5Ebov-10-Pan_2_LEFTCAAGCAAGATTGAGAATTAACCTTGGTEbov-10-Pan_2_RIGHTATCTCCCTGGTACGCATGATGA2100µM6Ebov-10-Pan_2_LEFT_alt1CAAGCAAGATTGAGAATTAACCTTGATEbov-10-Pan_2_RIGHT_alt1ATCTCCTTGGTACGCATGATGA2100µM7Ebov-10-Pan_3_LEFTGGCCTTTGAAGCAGGTGTTGATEbov-10-Pan_3_RIGHTTCAGTCCTTGCTCTGCATGTAC1100µM8Ebov-10-Pan_4_LEFTCCTTTGCAAGTCTATTCCTTCCGAEbov-10-Pan_4_RIGHTCTGAGTGCAGCCTTAAAGGAGT2100µM9Ebov-10-Pan_4_LEFT_alt1CTTTTGCAAGTCTATTCCTTCCGA2100µM10Ebov-10-Pan_5_LEFTAGTTCGTCTCCATCCTCTTGCAEbov-10-Pan_5_RIGHTCTGGAAGCTGATTTCGTTCTTTTTCT1100µM11Ebov-10-Pan_6_LEFTGAGTCTCGCGAACTTGACCATCEbov-10-Pan_6_RIGHTTCCTCGTCGTCCTCGTCTAGAT2100µM12Ebov-10-Pan_6_LEFT_alt1GAATCTCGCGAACTTGACCATCEbov-10-Pan_6_RIGHT_alt1TCCTCATCGTCCTCGTCTAGAT2100µM13Ebov-10-Pan_7_LEFTAGCTACGGCGAATACCAGAGTTEbov-10-Pan_7_RIGHTGTCCCTGTCCTGCTCTTCATCA1100µM14Ebov-10-Pan_7_RIGHT_alt1GTCCCTGTCCTGTTCTTCATCA1100µM15Ebov-10-Pan_7_RIGHT_alt2GTCCCTGTCCTGTTCTTCATCG1100µM16Ebov-10-Pan_8_LEFTTTAACGAAGAGGCAGACCCACTEbov-10-Pan_8_RIGHTTTCCTCTTCAAGGGAGTCTGGA2100µM17Ebov-10-Pan_8_LEFT_alt1TCAACGAAGAGGCAGACCCACTEbov-10-Pan_8_RIGHT_alt1TTCCTCTTCAAGGGAGTCCGGA2100µM18Ebov-10-Pan_9_LEFTGTGACAACACCCAGTCAGAACAEbov-10-Pan_9_RIGHTTCTTCCTGTTTTCGTTCCTTGACT1100µM19Ebov-10-Pan_9_LEFT_alt1GTGACAACACCCAGCCAGAACAEbov-10-Pan_9_RIGHT_alt1TCTTCCTGTTTGCGTTCCTTGACT1100µM20Ebov-10-Pan_9_RIGHT_alt2TCTTCCTGTTTGCGTTTCTTGACT1100µM21Ebov-10-Pan_10_LEFTACAATGGGATGATTCAACCGACAEbov-10-Pan_10_RIGHTTCGAGTGCTAGAGAATTCAATTGACG2100µM22Ebov-10-Pan_10_LEFT_alt1ATAATGGGATGATTTAACCGACA2100µM23Ebov-10-Pan_11_LEFTACCTACTAGCCTGCCCAACATTEbov-10-Pan_11_RIGHTAATTGGGTCCGTTTGGGTTTGA1100µM24Ebov-10-Pan_11_LEFT_alt1ACCTACTAGCCTACCCAACATTEbov-10-Pan_11_RIGHT_alt1AATTGGATCCGTTTGGGTTTGA1100µM25Ebov-10-Pan_12_LEFTCCCAAATGCAACAAACGAAGCCEbov-10-Pan_12_RIGHTTCAATCTTACCCCGAATCGCAC2100µM26Ebov-10-Pan_12_LEFT_alt1CCCAAATGCAACAAACAAAGCCEbov-10-Pan_12_RIGHT_alt1TCAATCTTACCCCGAATTGCAC2100µM27Ebov-10-Pan_13_LEFTTATTGGGCCGAACATGGTCAACEbov-10-Pan_13_RIGHTTGACAGGTGGAGCAGCATCTTG1100µM28Ebov-10-Pan_13_LEFT_alt1TATTGGGCTGAACATGGTCAAC1100µM29Ebov-10-Pan_14_LEFTCATTCATGCTGAGTTCCAGGCCEbov-10-Pan_14_RIGHTGCGAGATATGAACAATTTTATCTTGGTCG2100µM30Ebov-10-Pan_14_RIGHT_alt1GCGAGATAAGGACAATTTTATCTTGGTCG2100µM31Ebov-10-Pan_14_RIGHT_alt2GCGAGATAAGAACAATTTTATCTTGGTCG2100µM32Ebov-10-Pan_15_LEFTTGAGTATCAGCCCTGGATAATATAAGTCAEbov-10-Pan_15_RIGHTTCGATGGAGTGTCCCCATTGAC1100µM33Ebov-10-Pan_15_LEFT_alt1TGAGTATCAGCCCTAGATAATATAAGTCAEbov-10-Pan_15_RIGHT_alt1TCGATGGAGTGTCTCCATTGAC1100µM34Ebov-10-Pan_16_LEFTGCAACAGCAATACAGGCTTCCTEbov-10-Pan_16_RIGHTGAAAGCCTGGTTTCCAATTCGC2100µM35Ebov-10-Pan_16_LEFT_alt1GCAACAACAATACAGGCTTCCTEbov-10-Pan_16_RIGHT_alt1GAAGGCCTGGTTTCCAATTCGC2100µM36Ebov-10-Pan_17_LEFTCCACTTGTCAGAGTCAATCGGCEbov-10-Pan_17_RIGHTGTTTCTGGCACTTCGATTCCCA1100µM37Ebov-10-Pan_17_RIGHT_alt1GTTTCTGGCACTTCGATACCCA1100µM38Ebov-10-Pan_18_LEFTAAAATCCAAGCAATAATGACTTCACTCCEbov-10-Pan_18_RIGHTTTGATCAATTAAAAGTGTCTCCTCTAATGG2100µM39Ebov-10-Pan_18_RIGHT_alt1TCGATCAATTTAAAGTATCTCCTCTAATGG2100µM40Ebov-10-Pan_18_RIGHT_alt2TTGATCAATTAAAAGTATCTCCTCTAATAG2100µM41Ebov-10-Pan_19_LEFTAGATCCAGTTTTATAGAATCTTCTCAGGGAEbov-10-Pan_19_RIGHTAGAAGGGCAATGTCTGTACTTGG1100µM42Ebov-10-Pan_19_LEFT_alt1AGATCCAGTTTTACAGAATCTTCTCAGGGAEbov-10-Pan_19_RIGHT_alt1AGAAGGGCGATGTCTGTGCTTGG1100µM43Ebov-10-Pan_20_LEFTAGCCAGTGTGACTTGGATTGGAEbov-10-Pan_20_RIGHTAGTTTGTCGACATCACTAACCTGT2100µM44Ebov-10-Pan_20_RIGHT_alt1AGTTTGTCGACATCACTAACTTGT2100µM45Ebov-10-Pan_21_LEFTAGAACATTTTCCATCCCACTTGGAEbov-10-Pan_21_RIGHTAAGCACCCTCTTTATGGAAGGC1100µM46Ebov-10-Pan_21_RIGHT_alt1AAGCACCCTCTTTGTGGAAGGC1100µM47Ebov-10-Pan_22_LEFTTGCCGGTATGTGCACAAAGTATEbov-10-Pan_22_RIGHTATATATTGTCTCATTCAGCTGGAGCA2100µM48Ebov-10-Pan_23_LEFTCGAGGTTGACAATTTGACCTACGTEbov-10-Pan_23_RIGHTGCAAGGGTTGTTAGATGCGACA1100µM49Ebov-10-Pan_23_RIGHT_alt1GCAAGGGTTGTCAGATGCGACA1100µM50Ebov-10-Pan_24_LEFTTGCAATGGTTCAAGTGCACAGTEbov-10-Pan_24_RIGHTCTGGCACTCTCTTCTCCGGTAT2100µM51Ebov-10-Pan_24_LEFT_alt1TGCAATGGTTCAAGTGCACAAT2100µM52Ebov-10-Pan_25_LEFTACCACAACAAGTCCCCAAAACCEbov-10-Pan_25_RIGHTTAGCTCAGTTGTGGCTCTCAGG1100µM53Ebov-10-Pan_25_RIGHT_alt1TAGCTCGGTTGTGGCTCTCAGG1100µM54Ebov-10-Pan_26_LEFTATCTGTGGGTTGAGACAGCTGGEbov-10-Pan_26_RIGHTGCTTTTCCATGAAGCAATCTGAAGA2100µM55Ebov-10-Pan_26_LEFT_alt1ATCTGTGGATTGAGGCAGCTGGEbov-10-Pan_26_RIGHT_alt1GCTTTGCCATGAAGCAATCTGAAGA2100µM56Ebov-10-Pan_26_LEFT_alt2ATCTGTGGGTTGAGGCAGCTGG2100µM57Ebov-10-Pan_27_LEFTTGGAGTTACAGGCGTTATAATTGCAEbov-10-Pan_27_RIGHTAAAGGCTTCTTTCCCTTGTCACT1100µM58Ebov-10-Pan_28_LEFTTCATCCTTGATTCTACAATCATGACAGTEbov-10-Pan_28_RIGHTAGGTGCTGGAGGAACTGTTAATG2100µM59Ebov-10-Pan_28_LEFT_alt1TCATCCTTGATTCTACAATCATAACAGT2100µM60Ebov-10-Pan_29_LEFTGAGTACCGTCAATCAAGGAGCGEbov-10-Pan_29_RIGHTCACAGCACATAGAGTCAACAATGC1100µM61Ebov-10-Pan_30_LEFTGATCAAGACGGCAGAACACTGGEbov-10-Pan_30_RIGHTATCAGACCATGAGCATGTCCCC2100µM62Ebov-10-Pan_31_LEFTCTGCTGTCGTTGTTTCAGGGTTEbov-10-Pan_31_RIGHTATGGGATGGATCGTTGCTACCT1100µM63Ebov-10-Pan_31_RIGHT_alt1ATGGGATGGATCGTTGCTGCCT1100µM64Ebov-10-Pan_31_RIGHT_alt2ATGAGATGGATCGTTGCTACCT1100µM65Ebov-10-Pan_32_LEFTGCCAAGCATACCTCTTGCACAAEbov-10-Pan_32_RIGHTTGGACTACCCTGAAATAGTACTTTGC2100µM66Ebov-10-Pan_33_LEFTTGCGGAGGTCTGATAAGAATAAACCEbov-10-Pan_33_RIGHTTTCAACCTTGAAACCTTGCGCT1100µM67Ebov-10-Pan_33_RIGHT_alt1TTCAACTTTGAAACCTTGCGCT1100µM68Ebov-10-Pan_34_LEFTGCTGAAAAGAAGCTTACCTACAACGEbov-10-Pan_34_RIGHTTCCTTGTCATTGACCATGCAGG2100µM69Ebov-10-Pan_34_LEFT_alt1GTTGAAAAAAGGCCTACCTACAACG2100µM70Ebov-10-Pan_34_LEFT_alt2GCTGAAAAGAAGCCCACCTACAACG2100µM71Ebov-10-Pan_35_LEFTGTGACTCACAAAGGAATGGCCCEbov-10-Pan_35_RIGHTACAATCCGTTGTAGTTCACGACA1100µM72Ebov-10-Pan_35_RIGHT_alt1ACAACCCGTTGTAGTTCACGACA1100µM73Ebov-10-Pan_36_LEFTTGCTGTCGTTGATTCGATCCAAEbov-10-Pan_36_RIGHTAGCAGAGATGTCAAGATAACTATTGAGT2100µM74Ebov-10-Pan_37_LEFTACACGAATGCAAAGTTTGATTCTTGAEbov-10-Pan_37_RIGHTTGAAACCTAACACATGTGACCTGC1100µM75Ebov-10-Pan_37_RIGHT_alt1TGAAACCTAACACACGTGACCTGC1100µM76Ebov-10-Pan_38_LEFTCCCTCAAACAAGAGATTCCAAGACAEbov-10-Pan_38_RIGHTACAGTTGCGTAGTTGCGGATTA2100µM77Ebov-10-Pan_38_LEFT_alt1CCCTCAAATAAGAGATTCCAAGACA2100µM78Ebov-10-Pan_38_LEFT_alt2TCCTCAAATAAGAGATTCCAAGACA2100µM79Ebov-10-Pan_39_LEFTACCTAGTCACTAGAGCTTGCGGEbov-10-Pan_39_RIGHTACATTTGATGTAAAAATTCATTGCCCTG1100µM80Ebov-10-Pan_40_LEFTGTGGGTGCTCAAGAAGACTGTGEbov-10-Pan_40_RIGHTTGAGATTAGAGTTGTGTTGAATCGACA2100µM81Ebov-10-Pan_40_LEFT_alt1GTGGGTGCTCAAGAGGACTGTGEbov-10-Pan_40_RIGHT_alt1TGAGATTAGAGTCGTGTTGAATCGACA2100µM82Ebov-10-Pan_41_LEFTAAGAAGCGGTTCAAGGGCATACEbov-10-Pan_41_RIGHTCTATGGAATTCACGGATCTTTTGAGC1100µM83Ebov-10-Pan_41_LEFT_alt1AAGAAGCAGTTCAAGGGCATACEbov-10-Pan_41_RIGHT_alt1CTATGGAATTCACGGATCTTTTGATC1100µM84Ebov-10-Pan_42_LEFTTGCATTTAGCTGTAAATCACACCCTEbov-10-Pan_42_RIGHTAATCATTGGCAACGGAGGGAAT2100µM85Ebov-10-Pan_42_RIGHT_alt1AATCATTGGCAACGGGGGGAAT2100µM86Ebov-10-Pan_43_LEFTGTCAAGGATCTTGGTACAGTGTTACTEbov-10-Pan_43_RIGHTTGAGAAAGAAAAGTTCCGATATTGTGGT1100µM87Ebov-10-Pan_43_LEFT_alt1GCCAAGGGTCTTGGTACAGTGTTACTEbov-10-Pan_43_RIGHT_alt1TGAGAAAGAAAAATTCCGGTATTGTGGT1100µM88Ebov-10-Pan_43_LEFT_alt2GTCAAGGGTCTTGGTACAGTGTTACTEbov-10-Pan_43_RIGHT_alt2TGAGAAAGAAAAATTCCGATATTGTGGT1100µM89Ebov-10-Pan_44_LEFTTTGAGAATGTTCTTTCCTACGCACAEbov-10-Pan_44_RIGHTACGGTTGCAATATTCTATAAAAGGTGC2100µM90Ebov-10-Pan_44_LEFT_alt1TTGAGAATGTTCTTTCCTACGCGCAEbov-10-Pan_44_RIGHT_alt1ACGGTTGCAATATTCGATAAAAGGTGC2100µM91Ebov-10-Pan_44_RIGHT_alt2ACGGTTACAATATTCTATAAAAGGTGC2100µM92Ebov-10-Pan_45_LEFTCCACAGTTAGAGGGAGTAGCTTTGEbov-10-Pan_45_RIGHTGCTCGTCTGCGTCAGTCTCTAA1100µM93Ebov-10-Pan_45_LEFT_alt1CCACAGTTAGAGGGAGTAGTTTTG1100µM94Ebov-10-Pan_46_LEFTAAGTTACGCTCAGCTGTGATGGEbov-10-Pan_46_RIGHTATGGAAAGCTGCGGTTATCCTG2100µM95Ebov-10-Pan_47_LEFTTAGGCACTGCTTTTGAGCGATCEbov-10-Pan_47_RIGHTCACAAAGTCAATGGCAGTGCAG1100µM96Ebov-10-Pan_47_LEFT_alt1TAGGCACCGCTTTTGAGCGGTC1100µM97Ebov-10-Pan_47_LEFT_alt2TAGGCACTGCTTTTGAACGATC1100µM98Ebov-10-Pan_48_LEFTTCTCCGAATGATTGAGATGGATGATTEbov-10-Pan_48_RIGHTCTCAGTCTGTCCAAAACCGGTG2100µM99Ebov-10-Pan_48_LEFT_alt1TCTCCGAATGATTGGGATGGATGATT2100µM100Ebov-10-Pan_49_LEFTGATATCTTTTCACGCACGCCGAEbov-10-Pan_49_RIGHTCCACCTGGTTGCTTTGCATTTG1100µM101Ebov-10-Pan_49_LEFT_alt1GATATCTTTTCACGCACGCCCAEbov-10-Pan_49_RIGHT_alt1CCACCAGGTTGCTTTGCATTTG1100µM102Ebov-10-Pan_50_LEFTTCAAAGTGTTTTGGCTGAAACCCTEbov-10-Pan_50_RIGHTTCCTGAGTAATGTGAAGGGGTCA2100µM103Ebov-10-Pan_50_LEFT_alt1TCAAAGTGGTTTGGCTGAAACCCTEbov-10-Pan_50_RIGHT_alt1TCCTGAGTAATGTGAAGGAGTCA2100µM104Ebov-10-Pan_51_LEFTAACAGTGACTTGCTAATAAAACCATTTTTGEbov-10-Pan_51_RIGHTAAATACTGAGCTGGTACTTCCCG1100µM105Ebov-10-Pan_51_LEFT_alt1AACAGTGACTTGCTAATAAAGCCATTTTTG1100µM106Ebov-10-Pan_51_LEFT_alt2AACAGTGATTTGCTAATAAAACCATTTTTG1100µM107Ebov-10-Pan_52_LEFTAATCGTGCTCACCTTCATCTAACTEbov-10-Pan_52_RIGHTCCCAAAACTGTACAGAAGTCCTATCT2100µM108Ebov-10-Pan_53_LEFTACAGACCCAATTAGCAGTGGAGAEbov-10-Pan_53_RIGHTACAATTGTTCCGCGATTAATTATCCAT1100µM109Ebov-10-Pan_53_LEFT_alt1ACAGACCCAATTAGCAGCGGAGAEbov-10-Pan_53_RIGHT_alt1ACAATTGTTCCGCGATTAATTATCCACT1100µM110Ebov-10-Pan_54_LEFTTCTCAGATGCGGCCAGGTTATTEbov-10-Pan_54_RIGHTTGACCATCACTGTTGTTTGTGCT2100µM111Ebov-10-Pan_54_LEFT_alt1TCTCAGATGCGGCCAGATTATT2100µM112Ebov-10-Pan_55_LEFTTGGAGGAGCAGACACAGAAACAEbov-10-Pan_55_RIGHTATGACGTTAATTGGCGTGTCCC1100µM113Ebov-10-Pan_55_LEFT_alt1TGGAGGAGCAGGCACAGAAACAEbov-10-Pan_55_RIGHT_alt1ATGACGTCAATTGGCGTGTCCC1100µM114Ebov-10-Pan_55_LEFT_alt2TGGAGAAGCAGGCACAGAAACAEbov-10-Pan_55_RIGHT_alt2ATGACGTTAATTGGCGCGTCCC1100µM115Ebov-10-Pan_56_LEFTCTCACACCGTCTAGTCCTACCTEbov-10-Pan_56_RIGHTTTTGACATAACAGGTAGAAGCATCCT2100µM116Ebov-10-Pan_56_LEFT_alt1CTCGCACCGTCTAGTCCTACCT2100µM117Ebov-10-Pan_56_LEFT_alt2CTCACATCGTCTAGTCCTACCT2100µM118Ebov-10-Pan_57_LEFTACACGCTAGCTACTGAGTCCAGEbov-10-Pan_57_RIGHTATTGGCTTAATTAAATAACCAGTGGCA1100µM119Ebov-10-Pan_58_LEFTTGAAAGCAGTGGTCCTTAAAGTCTEbov-10-Pan_58_RIGHTTGCTCTAAGATGTGCTAAGTGCTG2100µM120Ebov-10-Pan_58_RIGHT_alt1TGCTCTAAGATGTGCCAAGTGCTG2100µM121Ebov-10-Pan_59_LEFTCGTCGATTCAAAAAGAGGTCCACTEbov-10-Pan_59_RIGHTTCAGAAGCCCTGTCAGCCTTTC1100µM122Ebov-10-Pan_60_LEFTAGATTGCAATTGTGAAGAACGTTTCTEbov-10-Pan_60_RIGHTAGAGTGCAGAGTTTATTATGTTGCGT2100µM123Ebov-10-Pan_61_LEFTTCACAATGCAGCATGTGTGACAEbov-10-Pan_61_RIGHTAGGTATTTCTGATTTTACAGTCCTGCC1100µM124Ebov-10-Pan_61_RIGHT_alt1AGGTATTTATGATTTTACAGTCCTGCC1100µM125Ebov-10-Pan_61_RIGHT_alt2AGGTATTTCTGATTTTACAGTCATGCC1100µM126Ebov-10-Pan_62_LEFTCCTGTCAGATGGAATAGTGTTTTGGTEbov-10-Pan_62_RIGHTAATTTTTGTGTGCGACCATTTTTCC2100µM
Primers need to be used at a final concentration of 0.015µM per primer. In this case, Pool 1 has 101 primers in it so the requirement is 3.8µL of Pool 1 (10µM) primers per 25µL reaction. Pool 2 has 106 primers the requirement is 4.0µL of Pool 2 (10µM) primers per 25µL reaction. For other schemes, adjust the volume added appropriately.
?. [Multiplex PCR]
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:Component                                             Pool 1                Pool 2 NEB Q5 Polymerase 2X MasterMix    Primer Pool 1 or 2 (10µM)                    Water                                                        Total
12.5 µl
12.5 µl
3.8 µl
4 µl
6.2 µl
6 µl
22.5 µl
22.5 µl
The mastermix hood should prepared by sterilising with UV and treated with MediPal wipes, DNAway and RNAseZap reagents. Wipe down the hood with each sequentially, allowing 5 minutes for drying between each. Pipettes should also be treated in the same way, and UV treated for 30 mins between library preparations.This step should be carried out in the mastermix hood and template should not be taken anywhere near the mastermix hood at any stage.
?. [Multiplex PCR]
In the template hood add  cDNA to each tube and mix well by pipetting.
2.5 µl
The template hood should prepared by sterilising with UV and treated with MediPal wipes, DNAway and RNAseZap reagents. Wipe down the hood with each sequentially, allowing 5 minutes for drying between each. Pipettes should also be treated in the same way, and UV treated for 30 mins between library preparations.
?. [Multiplex PCR]
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              25-35 Annealing                   25-35Hold                                   Indefinite              1
98 °C
98 °C
65 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35
?. [PCR clean-up]
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single Eppendorf tube.
1.5 ml
?. [Quantification and normalisation]
Quantify the amplicon pools using the Quantus Fluorometer using the ONE dsDNA assay.
?. [Quantification and normalisation]
Label a  Eppendorf tube for each sample.
1.5 ml
This is a ‘one-pot ligation’ protocol for native barcoded ligation libraries. We have seen no reduction in performance compared to standard libraries, and is made faster by using the Ultra II® ligation module which is compatible with the Ultra II® end repair/dA-tailing module removing a clean-up step.
?. [Quantification and normalisation]
Normalise the input but adding  amplicon pools to each tube and diluting to  to have an input concentration of
10 ng
10 µl
Quantity of amplicons can vary from 10-50ng depending on the amplicon length, any more than this and the molarity of DNA ends will be too high for efficient barcoding. You need to have 6 samples per native barcoded library to have sufficient material at the end.
?. [Native barocoding]
Barcode the amplicon pools using native barcodes.
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?. [Native barocoding]
Quantify the final library using the Quantus Fluorometer using the ONE dsDNA assay.
Final library can be now be stored in EB at 4°C for up to a week if needed otherwise proceed directly to MinION sequencing.
?. [MinION sequencing]
Start the sequencing run using MinKNOW.
?. [PCR clean-up]
Clean-up the amplicons using the following protocol:
?. [MinION sequencing]
Prime the flowcell and load  sequencing library onto the flowcell.
30 ng
From experience we know 30 ng is optimum loading input for short amplicons.