Bo Stevens AMF Library Prep

Plates: BS_PLT1, BS_PLT2

PCR MasterMix

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	390	1170
0.45	10M dNTPs	390	176
0.3	Kapa HiFi HotStart DNA Pol	390	117
7.25	HPLC H2O	390	2827
11	Total Volume	390	4290

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	Primer

Plate	Primer
BS_PLT1	AMF01
BS_PLT1	AMF02
BS_PLT2	AMF03
BS_PLT2	AMF04

Run 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

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 1,6,10 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	5	6	7	10	11	12
A	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool	NTC	NTC	NTC
B	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		0.02 pM Std	0.02 pM Std	0.02 pM Std
E	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		0.2 pM Std	0.2 pM Std	0.2 pM Std
F	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		2 pM Std	2 pM Std	2 pM Std
G	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		20 pM Std	20 pM Std	20 pM Std
H	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10

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	10	11	12
A	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool	NTC	NTC	NTC
B	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10	BS_AMF_Pool	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		0.02 pM Std	0.02 pM Std	0.02 pM Std
E	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		0.2 pM Std	0.2 pM Std	0.2 pM Std
F	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		2 pM Std	2 pM Std	2 pM Std
G	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10		20 pM Std	20 pM Std	20 pM Std
H	BS_PLT1_Col1	BS_PLT1_Col6	BS_PLT1_Col10	BS_PLT2_Col1	BS_PLT2_Col6	BS_PLT2_Col10

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)

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

Since the test sequencing included all samples, we are adjusting pooling for samples that both replicates returned less than 500 reads each. These samples will be pooled at a volume of 20 ul while all others will be pooled at 2 ul. 2 ul from all was combined into one column of strip tubes. 18 ul was added by well pattern above. Tubes were vortexed and centrifuged. 100 ul was added from each strip tube to a 1.5 ml tube which was vortexed and centrifuged before 40 ul was transferred to a tube for shipping.