scientificprotocols authored over 7 years ago
Authors: Rachel Graham
This protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of Bt-CoV HKU3 genomes.
This protocol details the steps, reagents, and conditions required to sequence Bt-CoV HKU3 genomes in the forward and reverse directions. The protocol begins with the RT-PCR step, assuming that BtCoV HKU3 RNA purified using a standard procedure (i.e., TRIzol extraction) has already been performed.
Reverse Transcription:
PCR (with Phusion PCR kit):
Annealing temperature is primer-dependent, but for most SARS-CoV primers in Tables 1 and 2, annealing temperatures 52-55ºC will work.
Confirmation of PCR products and sequencing:
Primers have been designed to give clean, single-band PCR products. If multiple bands are detected, alternate annealing temperatures may be required. It may also be possible that alternate bands indicate multiple genome patterns at that locus.
Table 1: Bt-CoV HKU3 Sequencing Primers. Bt-CoV HKU3 Sequencing Primers
1 BSStartF CCTACCCAGGAAAAGCCAAC
2 BS76F TAAAATCTGTGTGGCTGTCG
3 BS379F TTATCGGAGGCACGTGAACA
4 BS654F AGCCGGTGGTCATAGTTTTG
5 BS950F ACTGCTGCCGTGAACATGAA
6 BS1484F GCGGCTGTGTATTTTCCTAT
7 BS1904F TTTTCTCTCGCACACTGGAT
8 BS2464F AAGGCACCAAAAGAAGTCAC
9 BS3030F TTGCTCTTTCTACCCTCCTG
10 BS3475F ACAGTTGGAGGCTCATGTTT
11 BS4083F TAAGAAGTCGGGTGGGACTA
12 BS4429F GGTGTCCGGTTCTTCTTCTA
13 BS4909F AACCTCCACACGCATATTGT
14 BS5464F AAGGGTGTAGAGGCTGTGAT
15 BS6041F TAACCGTCACATTCTTCCCA
16 BS6557F CCGCTGCAATAAATAGTGTC
17 BS7023F TCAGGGTTATTTTCCCTGCA
18 BS7455F TGTCAATGGCGTGAAGAGAT
19 BS8058F TGGTGTCCTTTCTACATTCG
20 BS8509F CTTTTGTGTGTTCTTGCTGC
21 BS8916F CGTTTTGGCTGCTGAATGTA
22 BS9401F ACCATGTTGTTGCCGCTAAT
23 BS10049F ATGGTTTGTGGTTGGATGAC
24 BS10432F TGCGTGTCTTTCTGCTACAT
25 BS10842F GCCCTTTGACGTTGTTAGAC
26 BS11282F ATGATGCTGCTAGACGTGTG
27 BS11709F GTTGGGCATTGGAGGTAAAC
28 BS12142F TCTGAGTTTGACCATGATGC
29 BS12500F TTGTCCAGCTCAGTGAAATC
30 BS12975F GCTTTCTTTCTGTGCTTTCG
31 BS13469F CGAGAAAGTTGCTGGTTTTG
32 BS13924F AATTCTGCGATGCTATGCGT
33 BS14442F TCACGCCTCAGTTTTAAGGA
34 BS14948F GCGTAATGTCATCCCTACAA
35 BS15500F TGATAAGTACGTCCGCAATC
36 BS15941F AATTGACGCCTACCCACTTA
37 BS16475F AGCGACATGTGATTGGACTA
38 BS17016F CACTTTGCTATTGGACTTGC
39 BS17354F AGCTCAATTACCTGCACCAC
40 BS17897F CAAGCTGCAATTTACGAGTC
41 BS18453F CAAATGCTCAGTGATACGCT
42 BS18991F ACTGGAAATTCTACGACGCT
43 BS19461F GCTGGATTTAGCCTTTGGAT
44 BS19887F CTCATTGTCTTGTTTGACGG
45 BS20558F CCCAAAATTACAGGCAAGTC
46 BS20900F ATTGATTGGAGACTGTGCCA
47 BS21261F TGGAGGAACACAAACCCTAT
48 BS21550F CGCAACCTAAAATGGCACAA
49 BS21931F ATGCCTGGGTTTATCAGAGT
50 BS22485F TTCCCTTCTGTCTATGCATG
51 BS22889F CCCACCTGCTCTTAATTGTT
52 BS23404F AGCATGTCAACGCCTCTTAT
53 BS23816F GGACTTTGGCGGTTTCAATT
54 BS24103F GTGCAGCTCTTCAAATACCA
55 BS24664F CTCCAGCTATTTGTCACGAA
56 BS25073F GTATGTTTGGCTCGGCTTTA
57 BS25402F TTGTTGGCGTTGCACTTCTT
58 BS25992F CAAATACACACAATCGACGG
59 BS26434F ACTCCTGGAACAATGGAATC
60 BS26925F ACAAATTAGGAGCGTCGCAG
61 BS27500F GTGCAAGATCAGTTTCACCA
62 BS27920F TGGATCTAGAAAATCGGCTC
63 BS28445F GACGGCAAAATGAAAGAGCT
64 BS28861F TAAAGGCCAACAACAACCAG
65 BS29235F AAACATTCCCACCAACAGAG
66 BS29601F AAGAGCCACCACATTTTCAC
1 BS474R GAACACATAGGGCTGTTCAA
2 BS969R TTCATGTTCACGGCAGCAGT
3 BS1497R AAATACACAGCCGCCAAAAC
4 BS1923R ATCCAGTGTGCGAGAGAAAA
5 BS2438R TCTTTGCCACGAATACACTG
6 BS2820R CTTATCCACTCGCACATCAA
7 BS3471R AGGTCCATTTTGCCTGATGT
8 BS3636R AGCTGACAATAGAGGTGCAA
9 BS4113R TAGCATTTCCGTAGTCCCAC
10 BS4394R TTATACTTGCGCTGGATGGT
11 BS4927R CAATATGCGTGTGGAGGTTA
12 BS5480R ACAGCCTCTACACCCTTCAA
13 BS6060R TGGGAAGAATGTGACGGTTA
14 BS6577R GGACACTATTTATTGCAGCG
15 BS7040R CAGGGAAAATAACCCTGACA
16 BS7474R ATCTCTTCACGCCATTGACA
17 BS8077R CGAATGTAGAAAGGACACCA
18 BS8528R GCAGCAAGAACACACAAAAG
19 BS8935R TACATTCAGCAGCCAAAACG
20 BS9420R ATTAGCGGCAACAACATGGT
21 BS9975R AAAACCACTCTGCAAAACCG
22 BS10451R ATGTAGCAGAAAGACACGCA
23 BS10861R GTCTAACAACGTCAAAGGGC
24 BS11227R TAACACAGTCCTTAAGCCGA
25 BS11728R GTTTACCTCCAATGCCCAAC
26 BS12222R CTGCTTGTACATTTGGGTCA
27 BS12519R GATTTCACTGAGCTGGACAA
28 BS12994R CGAAAGCACAGAAAGAAAGC
29 BS13485R AACCAGCAACTTTCTCGTTG
30 BS13943R ACGCATAGCATCGCAGAATT
31 BS14461R TCCTTAAAACTGAGGCGTGA
32 BS14967R TTGTAGGGATGACATTACGC
33 BS15519R GATTGCGGACGTACTTATCA
34 BS15960R TAAGTGGGTAGGCGTCAATT
35 BS16494R TAGTCCAATCACATGTCGCT
36 BS17035R GCAAGTCCAATAGCAAAGTG
37 BS17180R ATCAAAACACTCTACACGCG
38 BS17630R ACCTATTTGTGGCCTGTTGA
39 BS17916R GACTCGTAAATTGCAGCTTG
40 BS18476R TTTCAGCGTATCACTGAGCA
41 BS18892R TGCTGTACTTTTCTGCATGC
42 BS19479R TCCAAAGGCTAAATCCAGCA
43 BS19906R CCGTCAAACAAGACAATGAG
44 BS20577R GACTTGCCTGTAATTTTGGG
45 BS20919R TGGCACAGTCTCCAATCAAT
46 BS21280R ATAGGGTTTGTGTTCCTCCA
47 BS21665R TGAGTCAAATGGCAGGAAGT
48 BS21950R ACTCTGATAAACCCAGGCAT
49 BS22417R TGAGTGGGTGAAACTCTGAA
50 BS22908R AACAATTAAGAGCAGGTGGG
51 BS23232R CACTAACACCACCAAAGGAA
52 BS23627R AGCCATTGAAACAGGCATCA
53 BS23835R AATTGAAACCGCCAAAGTCC
54 BS24122R TGGTATTTGAAGAGCTGCAC
55 BS24579R TAAGGTGGTAGCCTTTTCCA
56 BS25092R TAAAGCCGAGCCAAACATAC
57 BS25421R AAGAAGTGCAACGCCAACAA
58 BS25949R TGTAGCATTTTCAGCACCAG
59 BS26454R AGATTCCATTGTTCCAGGAG
60 BS26699R CAAACAGCCTGAAAGAAGCA
61 BS27301R GCGCTATCAATGTCAAGAAG
62 BS27939R GAGCCGATTTTCTAGATCCA
63 BS28465R GAGCTCTTTCATTTTGCCGT
64 BS28880R CTGGTTGTTGTTGGCCTTTA
65 BS29254R CTCTGTTGGTGGGAATGTTT
66 BS29620R GTGAAAATGTGGTGGCTCTT
67 BS29657R ATTCACTGTACCCTCGATCG
Table 2: Bt-CoV HKU3 Amplicon Primer Sets.
Source: Protocol Exchange. Originally published online 10 July 2014.