scientificprotocols authored over 6 years ago
Authors: Rachel Graham
This protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of Bt-CoV HKU5 genomes.
This protocol details the steps, reagents, and conditions required to sequence Bt-CoV HKU5 genomes in the forward and reverse directions. The protocol begins with the RT-PCR step, assuming that BtCoV HKU5 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 HKU5 Sequencing Primers
HKU5.5 Primers
HKU55 1F 25_F CCCTCTCTCTCGTTCTCTTG
HKU55 2F 587_F TACCAAAATGCAGCAGGAGA
HKU55 3F 1015_F GATTGTGTGGAATGTGCAAC
HKU55 4F 1576_F GCACTTTATTCCTCGTGCAA
HKU55 5F 2060_F ATCCCGTGTGATGTTACTGA
HKU55 6F 2530_F TGATCTCGTAATTCCTGGTG
HKU55 7F 2978_F GTGGATGTTGTGAAGGATGA
HKU55 8F 3598_F AATAAGTGACGATGTTCCCG
HKU55 9F 4010_F GTCAACTTTTATGGGCCACT
HKU55 10F 4497_F CTAAAGACTCATTGGCGGAC
HKU55 11F 4957_F CGATGGGGTTAATTTCAGGA
HKU55 12F 5460_F AGCTTACCACTCAAGCCAAA
HKU55 13F 5924_F GGGAATACTTGGGCTGATGT
HKU55 14F 6413_F CCAAAGGAGGATGTTAAACC
HKU55 15F 7046_F TCCACTTTACGCCTGCTTTT
HKU55 16F 7551_F TTACACACATCCCCTTGTTG
HKU55 17F 8087_F GATCGTGGCCAGGAAAATTT
HKU55 18F 8529_F ACCTTAACGCTGCTTCTGTT
HKU55 19F 9039_F TTACCGTGGCTGTTATTGCT
HKU55 20F 9482_F AATGGCTCTTGGGCTATCTA
HKU55 21F 10003_F TTGTAGTTTCCAGGATGCAG
HKU55 22F 10545_F TGGCTGTGGAATCTGCTAAC
HKU55 23F 10943_F CCAAATAAGACTGGAATCGC
HKU55 24F 11389_F TACGCTGTTTCTTTTGCCCA
HKU55 25F 11945_F TTTCGCTATCTCACGGGTAA
HKU55 26F 12544_F CGAGGATAAGAAGGCCAAGA
HKU55 27F 13010_F GTTGAGGGTCGTAAAATGCT
HKU55 28F 13543_F ACATGTTCGTGATCCTGTTG
HKU55 29F 14050_F GCTTTGAGGCACTTTGATCA
HKU55 30F 14536_F CAGCAGTATCACCCAAATTG
HKU55 31F 14959_F TCCTCAGTCACATTAAGGCA
HKU55 32F 15338_F GTGTTTCGATCCTTAGCACA
HKU55 33F 15866_F GCCAACCTGACCCTAAATTT
HKU55 34F 16461_F TGTCGGATCTTGTGTCGTGT
HKU55 35F 16936_F GAGCGTGAATTGTTGCTTGT
HKU55 36F 17450_F CACGCATTATACCTGCGAAA
HKU55 37F 17914_F TCTGCCATTAACAGACCTCA
HKU55 38F 18446_F CGATACCCAACTACCCAAAA
HKU55 39F 18906_F TTCGCCGTTACACTCTTATG
HKU55 40F 19466_F TCAATTTGCCTGGTTGCGAT
HKU55 41F 20070_F GTTTGTGCTCTGGGATTACG
HKU55 42F 20530_F GAGCATGTGGTTTATGGTGA
HKU55 43F 20946_F CATGCTGCCTAATTACAAGC
HKU55 44F 21434_F CATGGTGGACTGTGTTTTGC
HKU55 45F 22095_F TTGTTCGTATTGGAGCAGCT
HKU55 46F 22501_F TGCTGAGTGGTTTGGCATTA
HKU55 47F 23132_F GCTGGAGAAATTGTGCAGTT
HKU55 48F 23557_F TCATGGCATAACAGGAAGAG
HKU55 49F 24026_F CCTTTAACACTCGCACCAAT
HKU55 50F 24533_F CCACCCTTATATGACCCCTA
HKU55 51F 24943_F TGGCCGTCTTACATCTCTTA
HKU55 52F 25536_F AGTTGATGGTGCTGAGTGAA
HKU55 53F 25968_F GCTGGCATGTACACTAATCG
HKU55 54F 26510_F CTAAGCGCCGTTTTACTCCT
HKU55 55F 27000_F ACATTCTCACACCACCGAAT
HKU55 56F 27566_F TTGCAGGCGGGGTTGTTATA
HKU55 57F 28012_F CGGCTATTCGATTATGTGTG
HKU55 58F 28470_F TTCGGCTGTTTATGCGCACT
HKU55 59F 28928_F CAATGAAACCCCTACCAACA
HKU55 60F 29436_F AGAAACGGAAGAAGCAACAG
HKU55 61F 29990_F AAGTGGTGATGATGCTGCTA
HKU55 62F 30150_F CCCGATTCAGAGGCATAAAT
HKU55 1R 420_R CTTTCGCCGTCCATAAACCA
HKU55 2R 1024_R CCACACAATCCGGTACAATT
HKU55 3R 1593_R GCACGAGGAATAAAGTGCAT
HKU55 4R 2079_R TCAGTAACATCACACGGGAT
HKU55 5R 2549_R CACCAGGAATTACGAGATCA
HKU55 6R 2996_R CATCCTTCACAACATCCACA
HKU55 7R 3475_R CTTGGCAACTTCAGCATCAT
HKU55 8R 4028_R GTGGCCCATAAAAGTTGACA
HKU55 9R 4594_R TGCTAAGTCAAGCCCATGAG
HKU55 10R 4975_R CCTGAAATTAACCCCATCGA
HKU55 11R 5479_R TTTGGCTTGAGTGGTAAGCT
HKU55 12R 5942_R CATCAGCCCAAGTATTCCCA
HKU55 13R 6432_R GGTTTAACATCCTCCTTTGG
HKU55 14R 6935_R CAACTTTAACGGTGCTTTGG
HKU55 15R 7570_R CAACAAGGGGATGTGTGTAA
HKU55 16R 8109_R GCTAAATTTTCCTGGCCACG
HKU55 17R 8548_R AACAGAAGCAGCGTTAAGGT
HKU55 18R 9058_R AGCAATAACAGCCACGGTAA
HKU55 19R 9543_R TCACCACAATAGACACCAGG
HKU55 20R 10022_R CTGCATCCTGGAAACTACAA
HKU55 21R 10564_R GTTAGCAGATTCCACAGCCA
HKU55 22R 10962_R GCGATTCCAGTCTTATTTGG
HKU55 23R 11408_R TGGGCAAAAGAAACAGCGTA
HKU55 24R 11964_R TTACCCGTGAGATAGCGAAA
HKU55 25R 12563_R TCTTGGCCTTCTTATCCTCG
HKU55 26R 13028_R GCATTTTACGACCCTCAACA
HKU55 27R 13501_R TGTGACATCTGGGTGCTCTA
HKU55 28R 14070_R CTGATCAAAGTGCCTCAAAG
HKU55 29R 14552_R TTTGGGTGATACTGCTGATC
HKU55 30R 15050_R ATCGTTGGCAGGTTGTAAGC
HKU55 31R 15539_R ATAGCGCGATCACACTTAGG
HKU55 32R 15890_R TCCACAAATTTAGGGTCAGG
HKU55 33R 16473_R ACAAGATCCGACAGCCTGAA
HKU55 34R 16990_R GATTAAGTGGTGGTTTGGCT
HKU55 35R 17557_R ACGTTTCTGGCAGTGCATTA
HKU55 36R 17926_R TGTTAATGGCAGAGCTAGCA
HKU55 37R 18502_R TGACTTGCCTTATAGCCTCA
HKU55 38R 18930_R GCAGGCATAAGAGTGTAACG
HKU55 39R 19485_R ATCGCAACCAGGCAAATTGA
HKU55 40R 20090_R TCGTAATCCCAGAGCACAAA
HKU55 41R 20548_R CACCATAAACCACATGCTCA
HKU55 42R 20965_R GCTTGTAATTAGGCAGCATG
HKU55 43R 21450_R AAACACAGTCCACCATGCAA
HKU55 44R 21837_R TGAGACCGTAGACATGAACC
HKU55 45R 22468_R GAACGTGCAATTGATCAGGT
HKU55 46R 22896_R TCTTGAGTTGTTGCCTGTTC
HKU55 47R 23496_R TGCATGGGACAGACACTATT
HKU55 48R 24044_R TTGGTGCGAGTGTTAAAGGA
HKU55 49R 24445_R CATATAGCCAGGATCAGCAA
HKU55 50R 24958_R AGATGTAAGACGGCCATTGA
HKU55 51R 25455_R AATTCCTCCAGCTCATCCTT
HKU55 52R 25978_R TACATGCCAGCCGTATCAAA
HKU55 53R 26433_R GCTCGAAGGCTGTGCTATAA
HKU55 54R 27016_R CGGTGGTGTGAGAATGTTTA
HKU55 55R 27585_R TATAACAACCCCGCCTGCAA
HKU55 56R 28047_R AACCAGAAACACACTGCACA
HKU55 57R 28489_R AGTGCGCATAAACAGCCGAA
HKU55 58R 28982_R TCTGGGCTTAGTTCTTGGAC
HKU55 59R 29455_R CTGTTGCTTCTTCCGTTTCT
HKU55 60R 29913_R TGTAGTTGGGATTCTTTGGG
HKU55 61R 30391_R GCACTGTTCACTTGCAATCT
Table 2: SARS-CoV Sequencing Primers
Table 3: Bt-CoV HKU5 Amplicon Primer Sets
A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant S. Agnihothram, B. L. Yount, E. F. Donaldson, J. Huynh, V. D. Menachery, L. E. Gralinski, R. L. Graham, M. M. Becker, S. Tomar, T. D. Scobey, H. L. Osswald, A. Whitmore, R. Gopal, A. K. Ghosh, A. Mesecar, M. Zambon, M. Heise, M. R. Denison, and R. S. Baric, mBio 5 (2) e00047-14 - e00047-14 25/03/2014
Source: Protocol Exchange. Originally published online 10 July 2014.