Name | Secuencia 5'-3' | ARTIC Network V3 primers | |
ONT_Sseq_1_LEFT | TTTCTGTTGGTGCTGATATTGC ACAAAAGAAAATGACTCTAAAGAGGGTTT | nCoV-2019_70_LEFT | |
ONT_Sseq_1_RIGHT | ACTTGCCTGTCGCTCTATCTTC ACTCTGAACTCACTTTCCATCCAAC | nCoV-2019_72_RIGHT | |
ONT_Sseq_3_LEFT | TTTCTGTTGGTGCTGATATTGC AGAGTCCAACCAACAGAATCTATTGT | nCoV-2019_75_LEFT | |
ONT_Sseq_3_RIGHT | ACTTGCCTGTCGCTCTATCTTC ACCTGTGCCTGTTAAACCATTGA | nCoV-2019_76_RIGHT_alt0 | |
ONT_Sseq_5_LEFT | TTTCTGTTGGTGCTGATATTGC CAACTTACTCCTACTTGGCGTGT | nCoV-2019_78_LEFT | |
ONT_Sseq_5_RIGHT | ACTTGCCTGTCGCTCTATCTTC TGGAGCTAAGTTGTTTAACAAGCG | TGGAGCTAAGTTGTTTAACAAGCG | |
ONT_Sseq_7_LEFT | TTTCTGTTGGTGCTGATATTGC GGGCTATCATCTTATGTCCTTCCCT | nCoV-2019_82_LEFT | |
ONT_Sseq_7_RIGHT | ACTTGCCTGTCGCTCTATCTTC AGGTGTGAGTAAACTGTTACAAACAAC | nCoV-2019_84_RIGHT | |
ONT_Sseq_2_LEFT | TTTCTGTTGGTGCTGATATTGC ACACGTGGTGTTTATTACCCTGAC | nCoV-2019_72_LEFT | |
ONT_Sseq_2_RIGHT | ACTTGCCTGTCGCTCTATCTTC GCAACACAGTTGCTGATTCTCTTC | nCoV-2019_74_RIGHT | |
ONT_Sseq_4_LEFT | TTTCTGTTGGTGCTGATATTGC CCAGCAACTGTTTGTGGACCTA | nCoV-2019_77_LEFT | |
ONT_Sseq_4_RIGHT | ACTTGCCTGTCGCTCTATCTTC TGTGTACAAAAACTGCCATATTGCA | nCoV-2019_78_RIGHT | |
ONT_Sseq_6_LEFT | TTTCTGTTGGTGCTGATATTGC TTGCCTTGGTGATATTGCTGCT | nCoV-2019_80_LEFT | |
ONT_Sseq_6_RIGHT | ACTTGCCTGTCGCTCTATCTTC TGCCAGAGATGTCACCTAAATCAA | nCoV-2019_82_RIGHT | |
ONT_Sseq_8_LEFT | TTTCTGTTGGTGCTGATATTGC TGCTGTAGTTGTCTCAAGGGCT | nCoV-2019_84_LEFT | |
ONT_Sseq_8_RIGHT | ACTTGCCTGTCGCTCTATCTTC ACGAAAGCAAGAAAAAGAAGTACGC | nCoV-2019_86_RIGHT |
Step | Temperature (ºC) | Time | Cycles | |
Primer annealing | 25 | 2 min | 1 | |
cDNA Synthesis | 55 | 10 min | ||
Heat inactivation | 95 | 1 min | ||
Hold | 10 | - |
Reagent | PCR master mix pool A | PCR master mix pool B | |
Nuclease free water | 400 μL | 400 μL | |
Primer pool A (30 μM) | 25 μL | -- | |
Primer pool B (30 μM) | 25 μL | ||
Q5® Hot Start High-Fidelity 2X Master Mix | 625 μL | 625 μL | |
Final volume | 1050 μL | 1050 μL |
Step | Temperature (ºC) | Time | Cycles | |
Initial denaturation | 98 | 30 sec | 1 | |
Denaturation | 98 | 15 sec | 20 | |
Annealing and extension | 63 | 3 min | ||
Hold | 10 | -- | -- |
Reagent | Volume (µL) | |
Q5® Hot Start High-Fidelity 2X Master Mix | 6.00 | |
PCR Barcodes (BC01-BC96) | 1.00 | |
Final volume | 6.75 |
Step | Temperature (ºC) | Time | Cycles | |
Initial denaturation | 98 | 30 s | 1 | |
Denaturation | 98 | 15 s | 15 | |
Annealing | 62 | 15 s | ||
Extension | 72 | 1m | ||
Final extension | 72 | 10 m | 1 | |
Hold | 4 | -- | -- |
Sample/Reagent | Volume (µL) | |
Clean barcoded pool (1 µg) | 48 | |
NEBNext FFPE DNA Repair Buffer | 3.5 | |
NEBNext FFPE DNA Repair Mix | 2 | |
Ultra II End-prep reaction buffer | 3.5 | |
Ultra II End-prep enzyme mix | 3 | |
Final volume | 60 |
Step | Temperature (ºC) | Time | |
Enzymes incubation | 20 | 5 min | |
Enzymes inactivation | 65 | 5 min | |
Hold | 4 | -- |
Reagent | Volume (uL) | |
Clean end-prepped DNA | 60 | |
Ligation Buffer (LNB) | 25 | |
NEBNext Quick T4 DNA Ligase | 10 | |
AMX adapter | 5 | |
Final volume | 100 |
Sample/Reagent | Volume (uL) | |
Sequencing Buffer (SQB) | 37.5 | |
Loading Beads (LB) | 25.5 | |
DNA library (~300 ng) | 12 | |
Final volume | 75 |
Sample/Reagent | Volume (uL) | |
Sequencing Buffer (SQB) | 15 | |
Loading Beads (LB) | 10 | |
DNA library (~200 ng) | 5 | |
Final Volume | 30 |