Improved molecular typing assay for rhinovirus species

A, B and C

Yury A. Bochkov, Kristine Grindle, Fue Vang, Michael D. Evans, and James E. Gern

Online supplemental materials

Table of Contents

Supplementary Fig. S1. Neighbor-joining phylogenetic tree based on partial 5’-UTR nucleotide sequences of RV-A, RV-B and RV-C types (n = 156) constructed using MEGA software (MEGA 5.1). Supplementary Fig. S2. Cross-reactivity of novel “species-specific” primers targeting 5’-UTR.

Supplementary Fig. S3. Cross-reactivity of the novel “species-specific” primers targeting 5’- UTR is not completely eliminated by ExoSAP treatment of the first PCR products. Supplementary Fig. S4. Sensitivity of the original and modified PCR assays targeting 5’-UTR. Supplementary Fig. S5. Chromatograms generated by direct sequencing of the PCR product of RV-A57 clinical isolate using (A) original P3 or (B) new 5’UTR-revseq primer in SeqMan software (DNAStar, USA). Supplementary Fig. S6. Pairwise nucleotide p-distances between reference strains representing all known types of RV-A, RV-B and RV-C (intertype variability) for partial 5’UTR (A) and VP4/VP2 (B) regions calculated using MEGA software (MEGA 5.1). Supplementary Fig. S7. Neighbor-joining phylogenetic tree based on partial 5’UTR nucleotide sequences of RV-A, RV-B and RV-C types (n = 156) and W types (n = 59) constructed using MEGA software (MEGA 5.1). Supplementary Fig. S8. Neighbor-joining phylogenetic tree based on partial VP4/VP2 nucleotide sequences of RV-A, RV-B and RV-C types (n = 174) and W types (n = 58) constructed using MEGA software (MEGA 5.1). Supplementary Fig. S9. Summary of the clinical samples and RV isolates used for modification and validation of the molecular typing assay. Supplementary Tables 1–4. Supplementary Methods.

RV

-A11

RV

-A10

4 JX

1937

97R

V-A2

4

94

RV-

A90

91

RV-

A20

RV-

A68

97

81

RV-

A76

RV-

A33

RV-

A34

RV-

A18

RV-

A46

RV-A

08RV

-A95

99 RV-A

80RV

-A21

RV-A

50RV

-A59

RV-A6

3

99

87

RV-A10

RV-A31

RV-A47

99

RV-A10

0

RV-A56

RV-A66

RV-A77

74

98

RV-A57

RV-A105 JN614995

99

RV-A28

RV-A53

98

RV-A40

RV-A85

99

RV-A38

RV-A54

RV-A9899

RV-A25

RV-A6299

RV-A29

RV-A449978

94

RV-A39RV-A02RV-A4976

RV-A23RV-A30

83

RV-A60RV-A09RV-A32RV-A67

8796

93

RV-A15RV-A7499

70

RV-A55RV-A16RV-A81

9798

98

RV-A64RV-A94

97

RV-A82RV-A22

99

RV-A43RV-A75

93

RV-A01

88

RV-A19RV-A61

RV-A96

98

RV-A73

RV-A13

RV-A41

98

78

95

94

99

RV-A12

RV-A45

76

RV-A78

RV-A101 GQ415051

99

RV-C32 JN798581

RV-C39 JN205461

99RV-C13 HM

581812

75

RV-C43 JX074056

RV-C22 JN621242

RV-C

7 DQ

875932

RV-C

21 HM

581807

99R

V-C10 G

Q323774

RV-C

3 EF186077

RV-C

1 EF077279

RV-C

6 EF58238799

7499

99

RV-A07

RV-A88

90

RV

-A58

RV

-A3698

A-V

R29

9699

99

RV

-A65

RV-

A102

EF1

5542

199

RV-

A51

93

RV-

A103

JF9

6551

5

83

RV-

A71

RV-

A106

JX0

2555

5

99

99

RV-

C18

HM

5818

22

RV-

C28

HM

5818

59

84

RV-

C8

GQ

2232

27

73

RV-

C16

HM

5818

48

RV-C

17 H

M58

1840

99

99

RV-C

12 J

F317

017

RV-C

31 H

M58

1869

RV-C

42 J

Q99

4500

8699

99

RV-C

24 H

M58

1843

RV-C

25 JF

3170

13

99

RV-C15

GU21

9984

RV-C23

HM58

1805

87

RV-C30

HM58

1862

RV-C41

JN79

8565

99

RV-C2 E

F0772

80

RV-C40

JQ24

5963

RV-C5 EF582386

RV-C11 EU840952

RV-Cpat24 FJ200456

9999 99

95

RV-C4 EF582385

RV-C49 JN798566

99 99

RV-B17RV-B70 82RV-B91 99RV-B48RV-B52RV-B104 FJ445137

98 97RV-B69 99RV-B26RV-B42RV-B05

RV-B9973

95 99RV-B84RV-B86RV-B83RV-B92

75RV-B35RV-B79

99

RV-B04RV-B97

95

RV-B27

RV-B9386

RV-B03

RV-B37

RV-B06

RV-B103 JN798572

89

RV-B101 JF781501

RV-B102 JX074053

RV-B14

RV-B72

82

96

99

83

99

90

RV-C51 JF317015

RV-C51 JX291115

90

RV-C26 HM581808

99

RV-C36 JN541267

RV-C46 HM581884

79

98

RV-C9 GQ223228

RV-C14 HM581815

RV-C33 HM581838

99

RV-C50 JF317005

73

RV-C35 JF436925

RV-C19 EU840728

RV-C20 HM581870

RV-C27 HM581810

82

RV-C29 HM581853

RV-C45 JN837686

7199

9280

99

PV-1mC

V-A1399

PV-3l

99

90

0.00

0.05

0.10

0.15

Supplementary Fig. S1. Neighbor-joining phylogenetic tree based on partial 5’UTR nucleotide sequences of RV-A, RV-Band RV-C types (n=156) constructed using MEGA software (MEGA 5.1). All major nodes are labeled with bootstrap values(% of 500 replicates). RV-A and RV-B reference strain accession numbers correspond to those published previously(Palmenberg et al., 2009). Accession numbers of RV-C types and novel RV-A and RV-B types are shown. Branch lengthsare proportional to nucleotide similarity (p distance). Human enteroviruses (HEV) are included as an outgroup.

HEV

5’-UTR(~300 bp)

n=156 types

RV-ARV-Ca

RV-CcRV-B

L 1 2 3 4 5 6 7 8 9 10 L 11 12 13 14 15 16 17 18 19 20

Supplementary Fig. S2. Cross-reactivity of the redesigned forward primers targeting 5’-UTR. A total of 20 clinical samples(nasal secretions) containing 5 different types of RV-A (1-5), RV-B (6-10), RV-Ca (11-15) and RV-Cc (16-20) were testedby PCR using previously reported P1 and P3 primers in 1st reaction (A), and then reamplified using either the sameprimers, or novel 5’-UTRn-A, B and C primers, alone or combined in 2nd reaction (B-E). L is 1kb Plus DNA ladder (LifeTechnologies).

A

B

C

D

negative controls

RV-A RV-B RV-Ca RV-Cc

1 2 3 4 5 L 1 2 3 4 5 1 2 3 4 5 L 1 2 3 4 5 1 2 3 4 5 L

5’UTRn-AP1+P3 5’UTRn-B 5’UTRn-C 5’UTRn-A,B,C

6 7 8 9 10 L 6 7 8 9 10 6 7 8 9 10 L 6 7 8 9 10 6 7 8 9 L 10

11 12 13 14 15 L 11 12 13 14 15 11 12 13 14 15 L 11 12 13 14 15 11 12 13 14 15

E

RV species:Clinicalsamples:

Primers:Clinicalsamples:

16 17 18 19 20 L 16 17 18 19 20 16 17 18 19 20 L 16 17 18 19 20 16 17 18 19 20

L 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 L 1 2 3 4 5 1 2 3 4 5

5’UTRn-AP1+P3 5’UTRn-B 5’UTRn-C 5’UTRn-A,B,CPrimers:Clinicalsamples:

6 7 8 9 10 6 7 8 9 10 6 7 8 9 10 L 6 7 8 9 10 6 7 8 9 10

Supplementary Fig. S3. Cross-reactivity of the redesigned forward primers targeting 5’-UTR is not completelyeliminated by ExoSAP treatment of the 1st PCR products. RV-A (1-5) and RV-B (6-10) 1st PCR products (SupplementaryFig. 3A) were treated with exonuclease I and shrimp alkaline phosphatase (ExoSAP) mix, and then reamplified usingeither the same primers, or novel 5’-UTRn-A, B and C primers, alone or combined in 2nd reaction. L is 1kb Plus DNAladder (Life Technologies).

- Ctrls L A11 B97 C5 C26 A78 B6 A75 -

A

B

RV type:

PCR mix:(2nd rxn)

Platinum

L A11 B97 C5 C26 A78 B6 A75 -

GoTaq Green

- Ctrls L A11 B97 C5 C26 A78 B6 A75 - L A11 B97 C5 C26 A78 B6 A75 -

C

104 10-1 104 10-1 104 10-1 104 10-1

105 10-1 105 10-1 105 10-1 105 10-1

RV-

A16

RV-

B14

RV-

C15

P1+P3 5’UTRn-A,B,C+5’UTR-rev5’UTRn-A,B,C+5’UTR-rev

D

390 bp

390 bp

Supplementary Fig. S4. Sensitivity of the original and modified PCR assays targeting 5’-UTR. Second PCR products obtained using Platinum PCR Supermix, High Fidelity (Life Technologies) and GoTaq Green Master Mix (Promega) with original (A) or modified (B) assay. A total of seven RV positive samples containing RV-A, B or C clinical isolates and one RV negative sample (-) producing human sequence amplicon were tested. (C) Products obtained after the first PCR amplification using original or modified primers. (D) Re-amplification of the first PCR products obtained with original (left panel) or modified (right panel) primers using modified primers in second PCR. Serial 10-fold dilutions (from 104–105 PFU to 10-1 PFU) of representative strains of RV-A, B or C were tested. L is 1kb Plus DNA ladder (Life Technologies).

10 20 30 40 50 60 70 80 90 100 110

ATATGGCTTCACACCCGTAAGGGTGTGCAGGCAGCCACGCAGGCTAGAACACTGTCGCCAGNGGGGGTCTTCTAGCCTCATCTGCCAGGTCTACTGTGGGGTTCAGCGGAGCGA

120 130 140 150 160 170 180 190 200 210 220

CCAACCATAGGCTCTCAAAGATGGTACTAANCTTTGCGTAGGCACTTTGCGNATAACGATCTAAGTTGTTTTTGCCCTGTTGGAGCATATCAACTTTGACCGGTGA

B

A

Supplementary Fig. S5. Chromatograms generated by direct sequencing of the PCR product of RV-A57 clinical isolate using (A) original P3 or (B) new 5’UTR-revseq primer in SeqMan software (DNAStar, USA). Undetermined bases (N) are boxed.

10 20 30 40 50 60 70 80 90 100 110

ATATGGCTTCACACCCGTAAGGGTGTGCAGGCAGCCACGCAGGCTAGAACACTGTCGCCAGTGGGGGTCTTCTAGCCTCATCTGCCAGGTCTACTGTGGGGTTCAGCGGAGCGA

120 130 140 150 160 170 180 190 200 210 220

CCAACCATAGGCTCTCAAAGATGGTACTAAGCTTTGCGTAGGCACTTTGCGGATAACGATCTAAGTTGTTTTTGCCCTGTTGGAGCATATCAACTTTGACCGGGGA

RV-A (n=83)

0

50

100

150

200

250

300

0 0.05 0.1 0.15 0.2 0.25 0.3

RV-B (n=29)

0

10

20

30

40

50

60

0 0.05 0.1 0.15 0.2 0.25

Supplementary Figure S6. Pairwise nucleotide p-distances between reference strains representing all knowntypes of RV-A, RV-B and RV-C (intertype variability) for partial 5’UTR (A) and VP4/VP2 (B) regions calculated using MEGA software (MEGA 5.1).

5’UTR

RV-A (n=83)

0

100

200

300

400

500

600

700

0 0.05 0.1 0.15 0.2 0.25

RV-B (n=30)

0102030405060708090

0 0.05 0.1 0.15 0.2 0.25

VP4/VP2

nucleotide p-distance nucleotide p-distance

RV-C (n=64)

0

50

100

150

200

250

300

350

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

nucleotide p-distance

RV-C (n=47)

0102030405060708090

100

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

nucleotide p-distance

nucleotide p-distance nucleotide p-distance

A

B

RV

-A11

RV

-A10

4 JX

1937

97R

V-A

24

94

RV

-A90

89

RV

-A20

RV-A

6896

80

RV-A

76RV

-A33

RV-A

34RV

-A18

RV-A

46W

48 (R

V-A

*)

7078

RV-A

08RV

-A95

99 RV-A

21RV

-A80

90

RV-A50

W49

(RV-A

*)

RV-A59

RV-A63

99

80

RV-A57

RV-A105 JN

614995

99 RV-A39

RV-A15

RV-A74

99

99

99 W14 (RV-A68/C3)*

RV-C13 HM581812

W07 (RV-C13)99

RV-C32 JN798581

W58 (RV-C32)99

RV-C39 JN205461

W27 (RV-C39)99

99

RV-A45RV-A12

84

RV-A78RV-A101 GQ415051W28 (RV-A101)

9999

RV-C43 JX074056W36 (RV-C43)

99

RV-C21 HM581807W29 (RV-C21)

96

RV-C7 DQ875932

99

RV-C22 JN621242W15 (RV-C22)

99

78

RV-C3 EF186077W02 (RV-C3)

99

RV-C10 GQ323774

W55 (RV-C10)

99

RV-C1 EF077279

W56 (RV-C1)

99

RV-C6 EF582387

W24 (RV-C6)

85

81

99

78

99

76

97

71

W22 (RV-A*)

RV-A07

RV-A88

91

RV-A58

RV-A36

RV-A89

9496

84

99

99

RV-A65

RV-A102 EF155421

99RV-A51

93

RV-A103 JF965515

W06 (RV-A103)

99

91

RV-A

71

RV-A

106 JX025555

W53 (R

V-A

106)

99

9999

W47 (RV

-Cpat28)

W47-R

954 (RV

-C8)

82

RV-C8 G

Q223227

99

W59 (R

V-C

pat28)99

RV

-C18 H

M581822

W08 (R

V-C

18)99

RV

-C28 H

M581859

W20 (R

V-C

28)99

93

90

848185M

H 61C-

VR

W38

(RV

-C16

)99

RV

-C17

HM

5818

40

W57

(RV

-C17

)99

9999

RV

-C12

JF31

7017

W09

(RV

-C12

)

97

W04

(RV

-C44

)

99

RV-C

31 H

M58

1869

W30

(RV

-C31

)

99

RV-C

42 JQ

9945

00

W16

(RV

-C42

)99

80

99

99

RV-C

24 H

M58

1843

W34

(RV-

C24)

99

RV-C

25 JF

3170

13

W05

(RV-

C25)

99

99

RV-C

15 G

U2199

84

W10

(RV-C

15)

99

RV-C23

HM

5818

05

W17

(RV-C

23)

99

86

RV-C30

HM

5818

62

W42

(RV-C

30) 99

RV-C41

JN79

8565

* W25

(RV-C

41)

99

99

80

RV-C2 EF077280

W12 (RV-C2)

99

W39 (RV-C47)

RV-C40 JQ245963

W11 (RV-C40) 99

RV-C5 EF582386

W46 (RV-C5) 99

RV-C11 EU840952

RV-Cpat24 FJ200456W23 (RV-C11)

9999

98

99 95

RV-C4 EF582385RV-C49 JN798566

W33 (RV-C49)99

99

99

RV-B 85

RV-C51 JF317015W32 (RV-C37) 99

RV-C51 JX29111577

RV-C26 HM581808

W37 (RV-C26) 99

98

W26 (RV-Cpat10)

RV-C46 HM581884

W19 (RV-C46)

99

W52 (RV-Cpat21)

RV-C36 JN541267

W01 (RV-C36)99

99

80

97

RV-C9 GQ223228

W31 (RV-C9)99

RV-C33 HM581838

W54 (RV-C33)

98

W21 (RV-Cpat22)

98

RV-C14 HM581815

W03 (RV-C14)99

98

W51 (RV-Cpat22)

99

W43 (RV-C50)

W18 (RV-Cpat19)

RV-C35 JF436925

W40 (RV-C35)

99

99

88

RV-C19 EU840728

W41 (RV-C19)

99

RV-C27 HM581810

W44 (RV-C27)

99

RV-C29 HM581853

W45 (RV-C29)

99

W35 (RV-Cpat17)

83

RV-C20 HM581870

W50 (RV-C34)

99

RV-C45 JN837686

W13 (RV

-C45)99

7299

97

71

99

EV

85

0.00

0.05

0.10

0.15

5’-UTR(~300 bp)

RV-ARV-Ca

RV-Cc

R

R

R

R

Supplementary Fig. S7. Neighbor-joining phylogenetic tree based on partial 5’UTR nucleotide sequences of RV-A, RV-Band RV-C types (n=156) and Wisconsin (W) isolates (n=59) constructed using MEGA software (MEGA 5.1). All major nodesare labeled with bootstrap values (% of 500 replicates). All RV-B and some RV-A branches that do not contain novel types werecondensed for simplicity. RV-A reference strain accession numbers correspond to those published previously (Palmenberget al., 2009). Accession numbers of RV-C types and newly identified RV-A types are shown. Branch lengths are proportional tonucleotide similarity (p-distance). Human enteroviruses (HEV) are included as an outgroup. W types are followed bycorresponding RV-A or RV-C type designations based on VP4/VP2 partial sequences (http://www.picornastudygroup.com).Novel W types (W53-59) are shown in bold; putative recombinant W types (R) are underlined. Putative artificial recombinantW14, misidentified W25 (RV-C41) and three novel RV-A types (W22, W48 and W49) are labeled by star sign.

RV

-C2

W12

99

RV

-C40

W11

99

92

RV

-C47

W39

99

72

RV-C

pat

18RV

-C35

W40

99

RV-C

pat

19W

18

99

97

84

RV-C

14W

03

99

RV-C

33

W54

99 RV-C

48W

51RV-C

pat22

W21

(C33

)

98

99

7999

RV-C4

RV-C49

W33

99

99

89

RV-C41

W25

92

RV-C pat20

97

RV-C30

W42

99

99

RV-C19

W41

99 RV-C24

W34

99 RV-C25

W05

99

7990

RV-C23

W1799

RV-C51 JX291115

RV-C15W10

998999

RV-C12W0999

RV-C44W04

99 RV-C42W1699

95

RV-C16W38

99

RV-C17W5799

99

RV-C31W30

99RV-C pat28W47 (C8)W59

99

RV-C8W47-R954

99

99

RV-C18W08

99

RV-C pat27RV-C pat24

RV-C28W20

99

99

99

98

98

86

RV-C26W37

99

RV-C37W32 (C51)

99

81

RV-C51

RV-C51 JF317015

99

92

RV-C46W19

99RV-C36W

01

99RV-Cpat21

W52

99

9898

RV-C9W31

99RV-C50

W43

99RV

-C34

W50 (C20)

99RV

-C20

99RV

-C29W

4599

RV-C pat17

W35

99R

V-C

45W

1399

99

RV

-C27

W44

99

RV

-C5

W46

99

RV

-C38

RV

-C11

W23 (C

pat24)

9988

87

97

9985

75

RV-C

pat

10

W26

99

RV-C

1

W56

99

RV-C

pat

14

RV-C

pat

16

99

RV-C

3

W02

99

RV-C

6

W24

99

93

RV-C

10

W55

99

99

RV-C22

W15

99

RV-C13

W07

99

RV-C32

W58

99

RV-C39

W27

9999

RV-C43W36

99RV-C7RV-C21W29

9990 96

99 99

RV-A78RV-A65

RV-A102 EF15542192

RV-A51

95

RV-A71RV-A106 JX025555W53

99 94

RV-A103 JF965515W0699RV-A45

RV-A08RV-A95 99 96RV-A36RV-A58RV-A89

99

RV-A88W22

82

RV-A07

98

RV-A46

RV-A80

W48

97

RV-A53

RV-A101 GQ415051

W2899

RV-A28

70

RV-A31

RV-A47

99

W49

RV-A56

RV-A33

RV-A43

RV-A38RV-A60

9593

92

79

99

86

RV-B

EV

86

0.00

0.05

0.10

0.15

0.20

VP4-VP2(252-258 bp)

RV-A

RV-C

R

R

R

R

Supplementary Fig. S8. Neighbor-joining phylogenetic tree based on partial VP4/VP2 nucleotide sequences of RV-A, RV-Band RV-C types (n=174) and Wisconsin (W) isolates (n=58) constructed using MEGA software (MEGA 5.1). All major nodesare labeled with bootstrap values (% of 500 replicates). All RV-B and some RV-A branches that do not contain novel types werecondensed for simplicity. RV-A reference strain accession numbers correspond to those published previously (Palmenberget al., 2009), RV-C types are designated according to the proposed molecular classification by Simmonds et al, (2010) and McIntyre et all, (2013). Accession numbers of RV-C51 putative recombinant sequences and novel RV-A types are shown.Branch lengths are proportional to nucleotide similarity (p-distance). Human enteroviruses (HEV) are included as anoutgroup. The majority of W types cluster with corresponding RV-C reference types (http://www.picornastudygroup.com).Novel W types (W53-59) are shown in bold; putative recombinant W types (R) are underlined and followed by RV-C type(shown in parenthesis) that revealed the highest identity by 5’UTR partial sequence.

Supplementary Fig. S9. Summary of the clinical samples and RV isolates used for modification and validation of the molecular typing assay.

n = 3028 clinical samplesfrom 310 children

RV negative(n = 1913)

RV positive (37%)(n = 1115)

RV-A (66 types)n = 604

RV-B (16 types)n = 209

RV-C (42 types)n = 213

Multiple RV types(n = 89)

RhinoGen study samples

Viral sinusitis study samples

n = 785 clinical samplesfrom 142 children

RV negative(n = 619)

RV positive (21%)(n = 166)

RV-A (31 types)n = 92

RV-B (6 types)n = 10

RV-C (30 types)n = 62

Multiple RV types(n = 2)

Sample RV type Primer Length (bp) q>=40(q/bplength)x100

q>=30(q/bplength)x100

q>=20(q/bplength)x100

1 A57 P3 145 80 55 114 79 132 912 C6 P3 147 73 50 102 69 125 853 B35 P3 144 7 5 37 26 82 574 C46 P3 152 69 45 106 70 132 875 C44 P3 151 101 67 137 91 149 996 B83 P3 141 4 3 11 8 47 337 A75 P3 147 18 12 64 44 125 858 C15 P3 147 12 8 61 41 114 789 A59 P3 145 25 17 66 46 111 77

1 A57 5'UTR-revseq 145 142 98 143 99 144 992 C6 5'UTR-revseq 147 66 45 110 75 139 953 B35 5'UTR-revseq 144 89 62 131 91 144 1004 C46 5'UTR-revseq 152 146 96 152 100 152 1005 C44 5'UTR-revseq 152 31 20 72 47 116 766 B83 5'UTR-revseq 143 143 100 143 100 143 1007 A75 5'UTR-revseq 147 141 96 145 99 146 998 C15 5'UTR-revseq 147 134 91 145 99 147 1009 A59 5'UTR-revseq 145 105 72 135 93 141 97

Supplementary Table S1. Assessment of the quality of nucleotide sequences obtained by directsequencing of the PCR products of nine RV clinical isolates with P3 or 5’UTR-revseq primersimplemented by Phred. Quality scores (q) for each sequence fragment assigned to each base arelogarithmically linked to error probability.

Partial 5'UTR Partial VP4/VP2HRV type 1 HRV type 2 P-distance HRV type 1 HRV type 2 P-distance

1 RV-A08 RV-A95 0.00 1 RV-A08 RV-A95 0.012 RV-A29 RV-A44 0.02 2 RV-A29 RV-A44 0.053 RV-A25 RV-A62 0.02 3 RV-A25 RV-A62 0.074 RV-A65 RV-A102 0.03 4 RV-A31 RV-A47 0.075 RV-A54 RV-A98 0.03 5 RV-A20 RV-A68 0.096 RV-A31 RV-A47 0.04 6 RV-A65 RV-A102 0.107 RV-A59 RV-A63 0.04 7 RV-A24 RV-A104 0.108 RV-A11 RV-A104 0.05 8 RV-A23 RV-A30 0.109 RV-A15 RV-A74 0.05 1 RV-B05 RV-B99 0.10

10 RV-A32 RV-A67 0.05 2 RV-B17 RV-B91 0.1011 RV-A02 RV-A49 0.05 3 RV-B70 RV-B91 0.1012 RV-A20 RV-A68 0.05 1 RV-C28 RV-Cpat24 0.0613 RV-A24 RV-A104 0.05 2 RV-C11 RV-C38 0.0814 RV-A36 RV-A89 0.06 3 RV-C41 RV-Cpat20 0.1015 RV-A40 RV-A85 0.06 4 RV-C5 RV-C38 0.1016 RV-A08 RV-A21 0.06 5 RV-C29 RV-C45 0.1017 RV-A21 RV-A95 0.06 6 RV-C4 RV-C49 0.1018 RV-A02 RV-A30 0.06 7 RV-C29 RV-Cpat17 0.1019 RV-A29 RV-A62 0.06 8 RV-C5 RV-C11 0.1020 RV-A49 RV-A74 0.06 9 RV-C32 RV-C39 0.1021 RV-A09 RV-A32 0.0622 RV-A02 RV-A74 0.0623 RV-A47 RV-A66 0.0624 RV-A56 RV-A66 0.0625 RV-A21 RV-A54 0.061 RV-B52 RV-B104 0.022 RV-B17 RV-B70 0.033 RV-B17 RV-B91 0.034 RV-B70 RV-B91 0.045 RV-B03 RV-B37 0.056 RV-B04 RV-B97 0.057 RV-B03 RV-B06 0.058 RV-B03 RV-B103 0.059 RV-B06 RV-B37 0.05

10 RV-B06 RV-B103 0.0511 RV-B48 RV-B104 0.0612 RV-B48 RV-B52 0.0613 RV-B03 RV-B14 0.0614 RV-B83 RV-B92 0.0615 RV-B14 RV-B37 0.0616 RV-B14 RV-B72 0.0617 RV-B14 RV-B102 0.061 RV-C7 RV-C21 0.042 RV-C32 RV-C39 0.053 RV-C3 RV-C6 0.054 RV-C1 RV-C3 0.055 RV-C3 RV-C10 0.066 RV-C1 RV-C6 0.067 RV-C11 RV-Cpat24 0.068 RV-C5 RV-C11 0.069 RV-C6 RV-C10 0.06

10 RV-C1 RV-C10 0.06

# #

Supplementary Table S2. Pairwise nucleotide sequence comparisons of RV-A, B and C types that have < 7% p-distances in partial 5' UTR and / or ≤ 10% p-distances in VP4/VP2 typing regions.

5'U

TR n

ucle

otid

e p-

dist

ance

Viru

s ty

pe1

23

45

67

89

1011

1213

1415

1617

1819

2021

2223

2425

2627

2829

3031

3233

3435

3637

3839

4041

4243

1R

V-A

160.

320.

200.

170.

190.

210.

280.

190.

230.

280.

280.

370.

200.

220.

35n/

an/

a0.

240.

380.

32n/

a0.

25n/

a0.

340.

380.

370.

330.

350.

160.

300.

290.

260.

190.

240.

210.

290.

290.

190.

200.

280.

220.

300.

372

RV

-B14

0.39

0.34

0.34

0.34

0.34

0.34

0.36

0.36

0.35

0.34

0.36

0.35

0.35

0.35

n/a

n/a

0.35

0.36

0.37

n/a

0.39

n/a

0.34

0.36

0.36

0.33

0.35

0.34

0.36

0.35

0.37

0.33

0.35

0.35

0.34

0.34

0.35

0.34

0.33

0.35

0.35

0.38

3R

V-C

10.

390.

410.

050.

060.

120.

300.

060.

270.

300.

310.

380.

120.

180.

37n/

an/

a0.

170.

360.

32n/

a0.

29n/

a0.

360.

360.

380.

350.

360.

070.

320.

300.

290.

050.

170.

130.

300.

290.

060.

010.

300.

180.

290.

354

RV

-C3

0.40

0.43

0.17

0.05

0.12

0.31

0.06

0.25

0.29

0.29

0.37

0.12

0.18

0.35

n/a

n/a

0.19

0.37

0.33

n/a

0.26

n/a

0.34

0.37

0.37

0.34

0.35

0.02

0.30

0.30

0.26

0.04

0.19

0.12

0.31

0.30

0.05

0.05

0.29

0.18

0.30

0.35

5R

V-C

60.

400.

440.

170.

120.

100.

290.

060.

250.

290.

290.

370.

100.

180.

36n/

an/

a0.

160.

370.

32n/

a0.

27n/

a0.

360.

370.

370.

350.

360.

070.

320.

290.

270.

030.

160.

110.

290.

280.

060.

050.

290.

180.

300.

366

RV

-C7

0.43

0.41

0.20

0.22

0.23

0.29

0.11

0.27

0.29

0.33

0.36

0.04

0.20

0.36

n/a

n/a

0.19

0.39

0.34

n/a

0.28

n/a

0.34

0.39

0.37

0.32

0.35

0.13

0.33

0.30

0.27

0.10

0.19

0.04

0.29

0.29

0.12

0.11

0.32

0.20

0.29

0.34

7R

V-C

80.

420.

420.

300.

250.

300.

280.

290.

230.

230.

190.

370.

290.

290.

34n/

an/

a0.

290.

360.

32n/

a0.

25n/

a0.

320.

360.

360.

330.

330.

300.

230.

230.

240.

290.

290.

280.

020.

020.

290.

300.

190.

290.

080.

358

RV

-C10

0.39

0.41

0.16

0.15

0.17

0.21

0.27

0.25

0.30

0.29

0.38

0.11

0.17

0.37

n/a

n/a

0.17

0.37

0.32

n/a

0.25

n/a

0.36

0.37

0.39

0.35

0.37

0.07

0.30

0.30

0.25

0.06

0.17

0.12

0.28

0.28

0.01

0.06

0.28

0.17

0.29

0.34

9R

V-C

110.

390.

390.

330.

320.

330.

300.

310.

320.

230.

240.

350.

270.

270.

36n/

an/

a0.

270.

330.

28n/

a0.

06n/

a0.

330.

340.

360.

340.

340.

250.

230.

250.

060.

250.

270.

270.

220.

220.

250.

270.

240.

270.

240.

3510

RV

-C12

0.42

0.40

0.26

0.29

0.31

0.28

0.24

0.29

0.31

0.22

0.33

0.29

0.29

0.32

n/a

n/a

0.29

0.36

0.32

n/a

0.25

n/a

0.30

0.37

0.33

0.29

0.31

0.28

0.08

0.03

0.24

0.28

0.30

0.28

0.23

0.23

0.30

0.29

0.21

0.29

0.26

0.37

11R

V-C

170.

420.

430.

270.

250.

260.

290.

200.

270.

310.

240.

360.

310.

280.

36n/

an/

a0.

300.

340.

31n/

a0.

26n/

a0.

360.

340.

370.

330.

360.

300.

220.

230.

250.

290.

310.

300.

190.

180.

290.

310.

000.

280.

190.

3712

RV

-C20

0.41

0.42

0.33

0.31

0.30

0.32

0.31

0.33

0.19

0.31

0.31

0.35

0.38

0.22

n/a

n/a

0.38

0.24

0.25

n/a

0.37

n/a

0.20

0.25

0.04

0.21

0.20

0.37

0.35

0.34

0.36

0.38

0.39

0.34

0.37

0.37

0.38

0.38

0.36

0.38

0.38

0.32

13R

V-C

210.

410.

450.

230.

210.

210.

150.

290.

230.

320.

290.

290.

340.

190.

33n/

an/

a0.

190.

380.

33n/

a0.

27n/

a0.

330.

390.

370.

310.

340.

130.

320.

290.

260.

100.

190.

010.

290.

290.

110.

120.

310.

190.

300.

3414

RV

-C32

0.39

0.43

0.22

0.24

0.25

0.20

0.29

0.23

0.31

0.27

0.27

0.34

0.19

0.38

n/a

n/a

0.05

0.36

0.32

n/a

0.28

n/a

0.36

0.37

0.38

0.36

0.37

0.18

0.29

0.30

0.29

0.17

0.04

0.20

0.29

0.29

0.17

0.19

0.28

0.00

0.27

0.33

15R

V-C

330.

410.

400.

310.

300.

310.

300.

240.

300.

280.

250.

270.

260.

280.

26n/

an/

a0.

360.

230.

24n/

a0.

38n/

a0.

040.

230.

200.

110.

010.

350.

330.

320.

370.

360.

380.

330.

340.

340.

370.

370.

360.

380.

350.

3216

RV

-C34

0.41

0.44

0.35

0.29

0.30

0.34

0.32

0.34

0.18

0.30

0.30

0.11

0.33

0.34

0.27

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

17R

V-C

370.

400.

400.

270.

310.

270.

280.

300.

250.

290.

260.

290.

280.

280.

250.

250.

29n/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

a18

RV

-C39

0.41

0.42

0.20

0.24

0.24

0.23

0.29

0.24

0.32

0.29

0.26

0.34

0.21

0.10

0.26

0.34

0.25

0.36

0.33

n/a

0.28

n/a

0.36

0.37

0.38

0.36

0.35

0.19

0.28

0.29

0.28

0.17

0.01

0.19

0.28

0.28

0.17

0.17

0.29

0.05

0.27

0.35

19R

V-C

51_J

F317

015

0.37

0.40

0.27

0.25

0.26

0.26

0.29

0.23

0.28

0.24

0.25

0.24

0.28

0.25

0.24

0.26

0.15

0.25

0.10

n/a

0.35

n/a

0.21

0.01

0.24

0.21

0.22

0.37

0.38

0.38

0.34

0.37

0.37

0.37

0.36

0.35

0.37

0.37

0.33

0.36

0.33

0.35

20R

V-C

51_J

X29

1115

0.39

0.42

0.28

0.25

0.28

0.26

0.27

0.27

0.31

0.24

0.23

0.25

0.27

0.27

0.22

0.27

0.24

0.26

0.14

n/a

0.27

n/a

0.23

0.11

0.25

0.23

0.23

0.32

0.35

0.35

0.27

0.32

0.34

0.32

0.32

0.32

0.32

0.33

0.30

0.32

0.30

0.33

21R

V-C

pat2

20.

420.

420.

340.

300.

280.

290.

270.

290.

300.

280.

250.

240.

290.

290.

130.

250.

230.

270.

210.

21n/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

a22

RV

-Cpa

t24

0.41

0.41

0.25

0.25

0.25

0.23

0.19

0.30

0.32

0.27

0.19

0.29

0.25

0.27

0.26

0.31

0.29

0.27

0.26

0.25

0.25

n/a

0.36

0.35

0.37

0.35

0.36

0.25

0.26

0.27

0.02

0.27

0.29

0.27

0.25

0.25

0.25

0.29

0.25

0.28

0.23

0.35

23R

V-C

pat2

80.

400.

400.

260.

260.

270.

280.

130.

280.

310.

220.

170.

290.

290.

290.

250.

290.

270.

270.

270.

240.

270.

15n/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

an/

a24

W21

_(R

V-C

pat2

2)0.

430.

420.

330.

290.

280.

290.

260.

290.

300.

270.

250.

250.

280.

280.

120.

240.

230.

270.

210.

210.

020.

250.

270.

220.

200.

110.

040.

340.

320.

310.

350.

350.

370.

320.

330.

320.

360.

360.

360.

360.

330.

3225

W32

_(R

V-C

37)

0.39

0.41

0.26

0.30

0.27

0.28

0.30

0.24

0.29

0.26

0.27

0.29

0.27

0.25

0.23

0.29

0.02

0.24

0.14

0.23

0.22

0.29

0.25

0.22

0.24

0.22

0.22

0.37

0.39

0.39

0.34

0.38

0.37

0.37

0.36

0.36

0.37

0.37

0.34

0.37

0.33

0.36

26W

50_(

RV

-C34

)0.

420.

440.

340.

310.

280.

340.

340.

330.

170.

320.

300.

100.

330.

330.

260.

050.

280.

330.

270.

290.

240.

290.

310.

240.

270.

200.

190.

370.

350.

340.

370.

370.

400.

350.

360.

360.

380.

390.

370.

380.

370.

3127

W51

_(R

V-C

pat2

2)0.

420.

400.

320.

280.

290.

300.

260.

300.

310.

280.

260.

270.

280.

290.

130.

250.

230.

270.

230.

230.

050.

250.

270.

040.

230.

260.

120.

340.

320.

310.

330.

340.

370.

290.

320.

310.

350.

350.

320.

360.

320.

3228

W54

_(R

V-C

33)

0.42

0.40

0.31

0.29

0.30

0.29

0.24

0.30

0.29

0.26

0.26

0.25

0.28

0.26

0.00

0.26

0.25

0.26

0.24

0.21

0.13

0.26

0.25

0.12

0.24

0.26

0.12

0.35

0.32

0.31

0.35

0.36

0.37

0.33

0.33

0.32

0.36

0.36

0.35

0.37

0.33

0.31

29W

02_(

RV

-C3)

0.39

0.42

0.15

0.02

0.13

0.20

0.27

0.16

0.33

0.29

0.26

0.31

0.20

0.22

0.29

0.30

0.29

0.23

0.26

0.26

0.30

0.23

0.26

0.29

0.29

0.32

0.28

0.29

0.30

0.30

0.26

0.06

0.18

0.13

0.30

0.29

0.06

0.07

0.29

0.18

0.28

0.34

30W

04_(

RV

-C44

)0.

380.

390.

250.

250.

290.

280.

190.

260.

290.

160.

230.

310.

290.

270.

250.

290.

270.

270.

260.

250.

230.

240.

220.

220.

270.

310.

210.

250.

250.

060.

250.

300.

300.

310.

220.

230.

310.

320.

220.

290.

260.

3831

W09

_(R

V-C

12)

0.42

0.40

0.26

0.29

0.29

0.28

0.22

0.28

0.32

0.06

0.23

0.31

0.30

0.29

0.27

0.31

0.25

0.29

0.26

0.25

0.27

0.25

0.20

0.27

0.25

0.33

0.27

0.28

0.28

0.17

0.27

0.29

0.30

0.28

0.23

0.23

0.31

0.30

0.22

0.30

0.27

0.37

32W

23_(

RV

-C11

)0.

390.

390.

330.

320.

330.

300.

300.

310.

040.

300.

310.

180.

320.

300.

250.

170.

270.

320.

270.

310.

290.

310.

290.

280.

270.

170.

280.

250.

330.

290.

310.

270.

280.

260.

240.

240.

250.

290.

250.

290.

230.

3333

W24

_(R

V-C

6)0.

400.

440.

150.

120.

040.

230.

270.

170.

340.

300.

250.

300.

220.

260.

310.

300.

280.

250.

260.

270.

290.

230.

240.

290.

280.

300.

280.

300.

130.

280.

270.

340.

170.

100.

290.

280.

060.

050.

290.

170.

290.

3534

W27

_(R

V-C

39)

0.40

0.43

0.21

0.24

0.25

0.23

0.29

0.25

0.32

0.29

0.27

0.34

0.19

0.09

0.25

0.33

0.25

0.03

0.24

0.25

0.27

0.27

0.27

0.27

0.23

0.33

0.27

0.25

0.23

0.28

0.28

0.32

0.26

0.19

0.29

0.29

0.17

0.17

0.30

0.04

0.28

0.34

35W

29_(

RV

-C21

)0.

420.

440.

220.

190.

200.

120.

270.

210.

310.

310.

270.

330.

070.

210.

280.

320.

270.

210.

250.

260.

270.

250.

280.

260.

270.

310.

270.

280.

180.

250.

310.

310.

200.

200.

280.

280.

120.

120.

300.

200.

280.

3336

W47

_(R

V-C

pat2

8)0.

400.

390.

250.

260.

280.

280.

130.

280.

310.

230.

180.

300.

290.

290.

250.

290.

270.

270.

270.

250.

270.

150.

010.

270.

260.

300.

270.

260.

270.

210.

200.

300.

250.

270.

290.

010.

290.

300.

190.

290.

070.

3537

W47

-R95

4_(R

V-C

8)0.

400.

420.

270.

250.

290.

260.

060.

270.

300.

230.

190.

290.

300.

300.

250.

310.

290.

280.

270.

240.

250.

190.

120.

260.

290.

320.

270.

250.

260.

180.

210.

300.

270.

290.

280.

120.

290.

290.

180.

290.

070.

3538

W55

_(R

V-C

10)

0.38

0.42

0.16

0.15

0.15

0.22

0.28

0.03

0.32

0.27

0.27

0.33

0.23

0.23

0.29

0.33

0.25

0.23

0.22

0.28

0.29

0.29

0.28

0.29

0.23

0.32

0.30

0.30

0.16

0.26

0.29

0.31

0.16

0.24

0.22

0.28

0.27

0.06

0.28

0.17

0.28

0.34

39W

56_(

RV

-C1)

0.38

0.42

0.04

0.15

0.16

0.21

0.28

0.16

0.33

0.28

0.27

0.32

0.23

0.21

0.29

0.34

0.28

0.19

0.27

0.28

0.32

0.23

0.26

0.31

0.27

0.33

0.31

0.29

0.14

0.25

0.28

0.33

0.14

0.21

0.22

0.27

0.26

0.15

0.30

0.19

0.30

0.36

40W

57_(

RV

-C17

)0.

430.

430.

270.

250.

270.

290.

200.

270.

310.

250.

020.

310.

280.

270.

260.

310.

290.

270.

260.

230.

230.

200.

190.

230.

270.

310.

250.

250.

260.

230.

230.

310.

250.

280.

270.

190.

190.

260.

260.

280.

190.

3641

W58

_(R

V-C

32)

0.40

0.42

0.22

0.25

0.24

0.19

0.29

0.24

0.31

0.27

0.27

0.35

0.19

0.02

0.26

0.34

0.25

0.12

0.27

0.28

0.28

0.27

0.29

0.28

0.25

0.34

0.29

0.26

0.23

0.26

0.28

0.30

0.25

0.10

0.21

0.29

0.31

0.24

0.22

0.28

0.27

0.33

42W

59_(

RV

-Cpa

t28)

0.40

0.38

0.25

0.26

0.27

0.28

0.14

0.27

0.31

0.23

0.18

0.29

0.29

0.28

0.25

0.29

0.27

0.27

0.27

0.25

0.27

0.16

0.02

0.28

0.26

0.30

0.27

0.25

0.26

0.22

0.21

0.30

0.25

0.28

0.28

0.02

0.12

0.28

0.26

0.19

0.28

0.35

43P

V-3

l_K

0139

20.

410.

380.

400.

380.

380.

390.

380.

400.

410.

370.

370.

380.

390.

400.

390.

380.

380.

400.

370.

370.

350.

370.

360.

350.

380.

380.

370.

390.

380.

350.

340.

410.

380.

400.

380.

360.

380.

400.

410.

370.

400.

36VP

4/VP

2 nu

cleo

tide

p-di

stan

ce

Supp

lem

enta

ry T

able

S3.

. Pai

rwis

e nu

cleo

tide

sequ

ence

com

paris

ons

of s

elec

ted

RV

-A, B

and

C re

fere

nce

type

s an

d W

type

s th

at h

ave ≤

7% p

-dis

tanc

es in

par

tial 5

' UTR

and

≤ 1

0% p

-dis

tanc

es in

VP4

/VP2

typ

ing

regi

ons

(sho

wn

in

bold

).

W type (5'UTR)

RV-C type* (VP4/VP2)

5'UTRstnemmoCepytbus

W01 RV-C36 CcW02 RV-C3 CaW03 RV-C14 CcW04 RV-C44 CaW05 RV-C25 CaW06 RV-A103 n/aW07 RV-C13 CaW08 RV-C18 CaW09 RV-C12 CaW10 RV-C15 CaW11 RV-C40 CaW12 RV-C02 CaW13 RV-C45 CcW14 n/a n/a Putative artificial recombinant of RV-A68 and RV-C3W15 RV-C22 CaW16 RV-C42 CaW17 RV-C23 CaW18 RV-Cpat19 CcW19 RV-C46 CcW20 RV-C28 CaW21 RV-Cpat22 Cc Putative recombinant of RV-C33 (5'UTR) and RV-Cpat22 (VP4/VP2)W22 RV-A n/a Putative new RV-A type, 98% identity with FJ950948 (5'UTR) and 91% with KF034014 (VP4/VP2)W23 RV-C11 CaW24 RV-C6 CaW25 RV-C41 Ca Misidentified as RV-Cpat20 in Lee et al., (2012)W26 RV-Cpat10 CcW27 RV-C39 CaW28 RV-A101 n/aW29 RV-C21 CaW30 RV-C31 CaW31 RV-C09 CcW32 RV-C37 Cc Putative recombinant of RV-C51 (5'UTR) and RV-C37 (VP4/VP2)W33 RV-C49 CaW34 RV-C24 CaW35 RV-Cpat17 CcW36 RV-C43 CaW37 RV-C26 CcW38 RV-C16 CaW39 RV-C47 CaW40 RV-C35 CcW41 RV-C19 CcW42 RV-C30 CaW43 RV-C50 CcW44 RV-C27 CcW45 RV-C29 CcW46 RV-C05 CaW47 RV-Cpat28 Ca Putative recombinant of RV-C8 (5'UTR) and RV-Cpat28 (VP4-2)W47-R954 RV-C8 CaW48 RV-A n/a Putative new RV-A type, 99% identity with GQ223185 (5'UTR) and 98% with GQ223154 (VP4/VP2)W49 RV-A n/a Putative new RV-A type, 99% identity with GU933105 (5'UTR) and 99% with JX863815 (VP4/VP2)W50 RV-C34 Cc Putative recombinant of RV-C20 (5'UTR) and RV-C34 (VP4/VP2)W51 RV-Cpat22 CcW52 RV-Cpat21 CcW53 RV-A106 n/aW54 RV-C33 CcW55 RV-C10 CaW56 RV-C1 CaW57 RV-C17 CaW58 RV-C32 CaW59 RV-Cpat28 Ca

* RV-C types correspond to those proposed by Simmonds et al., (2010) and McIntyre et al., (2013).Putative recombinant W types are underlined, novel types are shown in bold, RV-A types are shown in red

Supplementary Table S4. Updated conversion of W types (5’UTR based classification) tocorresponding RV-C or novel RV-A types (VP4/VP2 based classification).

Supplementary methods 1. Total RNA extraction from clinical samples (nasal lavage fluid or sputum)

1. Make a stock of carrier mix (10 µl) for each sample:

a. 0.5 µl human DNA (100 ng/µl; Clontech, 639401), total = 50 ng b. 4 µl glycogen (5 µg/µl; Applied Biosystems, 9510), total = 20 µg c. 1 µl glycoblue (15 µg/µl, Applied Biosystems, 9515), total = 15 µg d. 4.5 µl PBS

2. Prepare 5× NA Extraction Buffer (500 ml): 125 ml 2M Tris, pH 7.5 -> 500 mM 150 ml 5M NaCl -> 1500 mM 5 ml 0.5M EDTA -> 5mM 220 ml ddH2O

3. To each 350 µl nasal sample (adjust volume with PBS if necessary),

a. Add 10 µl carrier mix and b. 25 µl 5×NA extraction buffer c. 0.75 mL Trizol LS (Invitrogen, 102960-28)

4. Mix well (shake 20 times) and then vortex for 10 minutes in thermomixer at max

speed at room temp.

5. Add 230 µl of chloroform (Sigma, C2432) to each sample.

6. Mix well (shake 20 times) and then vortex for 5 minutes in thermomixer at max speed at room temp.

7. Centrifuge at max speed (≥10,000 rpm) for 5 minutes at room temperature.

8. Add 0.6 mL µl isopropanol (Sigma, I9516; RNase-free) into a new tube.

9. Transfer the upper aqueous layer (~ 700 µl) from the sample tube to the

isopropanol tube. Try to avoid DNA carryover.

10. Mix well (shake 20 times), then incubate at room temp for ≥ 1 hour for RNA precipitation.

11. Centrifuge at max speed for 12 minutes at room temperature to pellet RNA

precipitate.

12. Remove supernatant by pipetting.

13. Add 0.7 mL of 75% ethanol to each tube and then mix well and vortex for 3 sec.

14. Centrifuge at max speed for 5 minutes at room temperature.

15. Remove supernatant by pipetting.

16. Air-dry pellets for ≥ 15 minutes at room temp.

17. Add 22 µl of Nuclease-Free water to pellet. Vortex vigorously to dissolve RNA.

18. Store RNA in -80°C freezer or proceed with RT reaction.

2. cDNA synthesis (RT): Reagents: High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor, 1000 reactions (Applied Biosystems, 4374967). This kit contains 10× RT buffer, 25× dNTP mix, 10× RT random primers, MultiScribe RT enzyme and RNase inhibitor. Procedure: 1. Prepare RT master mix: For 1 reaction: 4 µl 10× RT buffer 1.6 µl 25× dNTP mix 4 µl 10× RT random primers 2 µl MultiScribe RT 2 µl RNase inhibitor 6.4 µl water 2. Aliquot 20 µl of RT master mix into each tube. 3. Add 20 µl of RNA into tubes with master mix, pipet up and down 2 times to mix, and centrifuge briefly. Run the RT reaction. Reaction conditions: 25°C, 10 min, 37°C 120 min, 85°C, 5 min.