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Gregg Randolph

Gregg Randolph

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Plates: BS_PLT1, BS_PLT22AMF1 and 2AMF2

 

1

2

3

4

5

6

7

8

9

10

11

12

1

A

2

23

34

44

54

63

71

92

 185

193

201

209

216

B

6

24

36

45

55

64

72

178 

186 

194

202

210

217

C

15

26

38

46

56

65

85

179 

187 

195

203

211

218

D

16

28

39

47

57

66

86

180 

188 

196

204

212

219

E

19

29

40

48

58

67

87

181 

189 

197

205

213

220

F

20

30

41

50

59

68

88

182 

190 

198

206

214

221

G

21

32

42

51

61

69

90

183 

191 

199

207

215

222

H

22

33

43

52

62

70

91

184 

192 

200

208

Blank

Blank2

Remaining 2 columns are Mock Community

PCR MasterMix

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

390530

11701590

0.45

10M dNTPs

390530

176238

0.3

Kapa HiFi HotStart DNA Pol

390530

117159

7.25

HPLC H2O

390530

28273843

11

Total Volume

390530

42905830

  • Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.

BS_PLT1BS_PLT2

Plate

18S Primer

ITS

2AMF1

AMF01

ITS0A9

AMF02

ITS0B9

AMF03

2AMF2

AMF04

ITS0C9

Run AMF on thermocycler program AMF35:

Step

Temp C

Cycles

Time

Denature

95

1X

10:00

Denature

95

35X

0:30

Annealing

55

35X

0:30

Extension/Elongation

72

35X

1:00

Extension/Elongation

72

1X

9:00

Hold

4

1X

0:00

And ITS on GSAF36

MagBead Cleanup:

  • Equilibrate Beads to room temperature

  • Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of replicate

  • Pipette mix up and down 10 times.

  • Incubate at RT for 5 minutes

  • Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

  • Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

  • Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

  • Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well

  • Reaspirate from each well to assure maximum EtOH removal

  • Allow plate to air dry for 7 minutes.

  • Remove sample plate from magnet plate.

  • Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.

  • Place sample plate back on magnet for 5 minutes or until all wells are cleared.

  • Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)

...

  • Make 1:1000 dilutions of column 1,6,10 4 or 2 and 4 (2AMF2) from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

 

1

2

3

4

5

6

7

8

9

10

11

12

A

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10

AMF

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

BS_AMF_Pool

2AMF2_ITS

 

NTC

NTC

NTC

B

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10

AMF

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

 BS_AMF_Pool

2AMF2_ITS

 

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10

AMF

BS

2AMF2_

PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

 BS_AMF_Pool

ITS

 

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10

AMF

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

2AMF2_ITS

 

 

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10

AMF

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

2AMF2_ITS

 

 

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10

AMF

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

2AMF2_ITS

 

 

 

2 pM Std

2 pM Std

2 pM Std

G

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10BS_PLT2_Col10

AMF

BS_PLT2_Col1

BS_PLT2_Col6

2AMF2_ITS

 

 

 

20 pM Std

20 pM Std

20 pM Std

H

BS

2AMF1_

PLT1_Col1

AMF

BS

2AMF1_

PLT1_Col6

ITS

BS

2AMF2_

PLT1_Col10

AMF

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

2AMF2_ITS

 

 

 

 

 

 

  • Add 16 ul of Illumina Library Quantification MasterMix to each well:

...

  • Add 4 ul of template, pool, or standards to each well:

 

1

2

3

4

5

6

7

8

9

10

11

12

A

BS2AMF1_PLT1AMF

2AMF1_Col1ITS

BS2AMF2_PLT1_Col6AMFBS

2TRNL_PLT1_Col10

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

BS_AMF_Pool

 

NTC16S

40 16S

40 16S

40 16S

40 ITS

40 ITS

40 ITS

NTC

NTC

B

BS2AMF1_PLT1_Col1AMFBS

2AMF1_PLT1_Col6ITSBS

2AMF2_PLT1_Col10AMFBS

2TRNL_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

BS_AMF_Pool

 

 

0.0002 pM Std16S

4 16S

4 16S

4 16S

4 ITS

4 ITS

4 ITS

0.0002 pM Std

0.0002 pM Std

C

BS2AMF1_PLT1_Col1AMFBS

2AMF1_PLT1_Col6ITSBS

2AMF2_PLT1_Col10AMFBS

2TRNL_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

BS_AMF_Pool

 

 

0.002 pM Std16S

0.4 16S

0.4 16S

0.4 16S

0.4 ITS

0.4 ITS

0.4 ITS

0.002 pM Std

0.002 pM Std

D

BS2AMF1_PLT1_Col1AMFBS

2AMF1_PLT1_Col6ITSBS

2AMF2_PLT1_Col10AMFBS

2TRNL_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

 

 

 

0.02 pM StdT

0.04 16S

0.04 16S

0.04 16S

0.04 ITS

0.04 ITS

0.04 ITS

0.02 pM Std

0.02 pM Std

E

BS2AMF1_PLT1_Col1AMFBS

2AMF1_PLT1_Col6ITSBS

2AMF2_PLT1_Col10ITSBS

2TRNL_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

 

 

 

0.2 pM StdT

0.004 16S

0.004 16S

0.004 16S

0.004 ITS

0.004 ITS

0.004 ITS

0.2 pM Std

0.2 pM Std

F

BS2AMF1_PLT1_Col1AMFBS

2AMF1_PLT1_Col6ITSBS

2AMF2_PLT1_Col10ITSBS

2TRNL_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

 

 

 

2 pM StdT

0.0004 16S

0.0004 16S

0.0004 16S

0.0004 ITS

0.0004 ITS

0.0004 ITS

2 pM Std

2 pM Std

G

BS2AMF1_PLT1_Col1AMFBS

2AMF1_PLT1_Col6ITS

BS_PLT1_Col10

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col10

2AMF2_ITS

 

 

 

20 pM Std

20 pM Std

20 pM Std

H

BS2AMF1_PLT1_Col1AMFBS

2AMF1_PLT1_Col6ITS

BS_PLT1_Col10

BS_PLT2_Col1

BS_PLT2_Col6

BS_PLT2_Col102AMF2_ITS

 

 

 

 

 

 

Results:

View file
nameBS_AMF.csv

...

Code Block
library(tidyverse)
AmfReads <- read.csv("/Users/gregg/Downloads/AMF1_Test_filtermergestats.csv", header = FALSE)
Map <- read.csv("/Users/gregg/Downloads/Bo_Stevens_Sample_Positions.csv", header=TRUE)
Map <- Map[,c(1,2,4)]
Map$order <- c(1:96, 1:92)
AMFmeta <- data.frame(do.call('rbind', strsplit(as.character(AmfReads$V1),'.',fixed=TRUE)))
AmfReads <- cbind(AMFmeta$X2, AmfReads[,2:4])
names(AmfReads) <- c("Sample.ID", "Reads", "Reads2", "Reads3")
AmfReads <- inner_join(AmfReads, Map, by = "Sample.ID")
LowAmf <- AmfReads[ which(AmfReads$Reads < 500),]
write.csv(LowAmf, "/Users/gregg/Downloads/LowAmf.csv", quote = FALSE, row.names = FALSE)
AMF1_Test_filtermergestats.csv Bo_Stevens_Sample_Positions.csv
Sample.ID
Reads
Reads2
Reads3
Plate.Position
Plate
order
A26
35
26
18
A9
2
65
A27
42
20
15
B9
2
66
A29
16
7
4
C9
2
68
A30
36
24
11
D9
2
69
A31
40
20
15
E9
2
70
A32
28
18
11
F9
2
71
A34
36
12
8
G9
2
73
A35
34
18
10
H9
2
74
B3
392
248
149
G11
2
91
L108
77
47
32
F4
1
30
L54
70
33
19
F7
1
54
L88
416
236
143
A4
1
25
LB1
104
51
34
A2
1
9
LB2
164
107
45
F3
1
22
LB3
70
35
20
C5
1
35
LB4
308
194
159
H6
1
48
LB5
310
169
129
E8
1
61
LB6
118
68
50
B10
1
74
LB7
374
219
161
G11
1
87
...