2AMF Library Prep

Plates: 2AMF1 and 2AMF2

	1	2	3	4	5	6	7	8	9	10	11	12	1

	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

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	530	1590
0.45	10M dNTPs	530	238
0.3	Kapa HiFi HotStart DNA Pol	530	159
7.25	HPLC H2O	530	3843
11	Total Volume	530	5830

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.

Plate	18S Primer	ITS

Plate	18S Primer	ITS
2AMF1	AMF01	ITS0A9
2AMF1	AMF02	ITS0B9
2AMF2	AMF04	ITS0C9
2AMF2

Run AMF on thermocycler program AMF35:

Step	Temp C	Cycles	Time

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)

qPCR

Make 1:1000 dilutions of column 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

	1	2	3	4	10	11	12
A	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS	NTC	NTC	NTC
B	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS	2 pM Std	2 pM Std	2 pM Std
G	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS	20 pM Std	20 pM Std	20 pM Std
H	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2AMF2_ITS

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

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	110	1100
2 ul	Primer Premix (10X)	110	220
4 ul	Ultra Pure Water	110	440
16 ul	Total Volume	110	1760

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

	1	2	3	4	5	6	7	8	9	10	11	12

	1	2	3	4	5	6	7	8	9	10	11	12
A	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2TRNL_16S	40 16S	40 16S	40 16S	40 ITS	40 ITS	40 ITS	NTC	NTC
B	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2TRNL_16S	4 16S	4 16S	4 16S	4 ITS	4 ITS	4 ITS	0.0002 pM Std	0.0002 pM Std
C	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2TRNL_16S	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	2AMF1_AMF	2AMF1_ITS	2AMF2_AMF	2TRNL_T	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	2AMF1_AMF	2AMF1_ITS	2AMF2_ITS	2TRNL_T	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	2AMF1_AMF	2AMF1_ITS	2AMF2_ITS	2TRNL_T	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	2AMF1_AMF	2AMF1_ITS	2AMF2_ITS								20 pM Std	20 pM Std
H	2AMF1_AMF	2AMF1_ITS	2AMF2_ITS

Results:

There were some outliers, but most samples generated 10s of nano moles (~50).

The pool via standard size estimation returned a mean of 42.66 nM. This should be adjusted for the difference between the standards' fragment sizes and the expected product size (452 vs 481). 42.66x(452/481) = 40.087 or ~40 nM

Sequencing Test:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

1000/Results = ul of Pool to Add

1000/40 = 25uL of Pool to Add

1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

1000- 25 = 975 uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 50 pM:

Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX
Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.
Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100
Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.

Pooling for CU sequencing:

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)

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