Low Read Experiment II

16S0C4 and 16S0D4 were used.

Gregg Randolph Linda van Diepen Alex Buerkle

This experiment is being repeated due to some errors that occurred during the initial experiment.

Questions:

How can we best accommodate low concentration input DNAs?
1. Concentrate input DNAs?
2. Concentrate/normalize low yield products?
3. Both?
4. Just put more volume of original DNA in?
In the sequence yield, does input concentration, substrate, or some other factor correlate well with low read counts?

Investigations:

Bench(Question 1):

Experimental Sketch of crossed design:

Pull aliquots of some high read count and low read count samples. (Pending new low read samples).
Quantify all samples not including blanks or mock community.
For previously low yielding samples, repeat at typical template amount, and at 2x, and 4x volume added to the reaction (in column 2,3,4, and 6, 7, 8). 6x8 samples in low yield, 8 in high yield, 8 of mock community, 8 of blank (ISD only). 72 reactions with different templates. See template below for clarification.
Template layout is replicated/duplicated, with different barcodes for duplicates.
1 set of 36 products will not be adjusted for pooling.
1 set of 36 will be adjusted for pooling per qPCR results.

Start Reaction Plate Setup:

Add Ultrapure H2O to the reaction plates in the following pattern:

NOTE: Water in the master mix has been adjusted to a lower amount. The plate set up below will replace the water usually added to the master mix while also showing if a decrease in water and an increase in template will increase PCR yields.

	1	2	3	5	6	7	9
A	6ul	6ul	4ul	6ul	6ul	4ul	6ul
B	6ul	6ul	4ul	6ul	6ul	4ul	6ul
C	6ul	6ul	4ul	6ul	6ul	4ul	6ul
D	6ul	6ul	4ul	6ul	6ul	4ul	6ul
E	6ul	6ul	4ul	6ul	6ul	4ul	6ul
F	6ul	6ul	4ul	6ul	6ul	4ul	6ul
G	6ul	6ul	4ul	6ul	6ul	4ul	6ul
H	6ul	6ul	4ul	6ul	6ul	4ul	6ul

MasterMix

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	200	600
0.45	10M dNTPs	200	90
0.3	Kapa HiFi HotStart DNA Pol	200	60
0.25	HPLC H2O	200	50
1	ISD	200	200
5	Total Volume	200	1000

Add 5 ul to each well of a hard shell, full skirt plate.
Add Templates following the pattern below:

Sample	Status	Plate	Location	Original Plate Name	DNA Quant ng/uL (>10ng/uL will be normalized)
`SAG_S30P2R`	Low Read	PA13	G6	AYAYEE_RHIZO_PLATE2	4.166
`SAG_S38P1R`	Low Read	PA13	B9	AYAYEE_RHIZO_PLATE2	9.122
`SAG_S15P4R`	Low Read	PA13	B2	AYAYEE_RHIZO_PLATE2	3.69
`SAG190005`	Low Read			Tube 33	29.422
`SAG192199`	Low Read			Tube 123	42.604
`SAG192077`	Low Read			Tube 96	2.469
`SAG190271`	Low Read			Tube 258	12.88
`SAG190238`	Low Read			Tube 255	34.242
`SAG192203`	Avg Read	PA2	F9	Tube 127	8.333
`SAG191508`	Avg Read	PA1	A12	Tube 73	32.279
`SAG_S16P3R`	Avg Read	PA12	G3	AYAYEE_RHIZO_PLATE1	2.885
`SAG_S44P2R`	Avg Read	PA12	F7	AYAYEE_RHIZO_PLATE1	6.397

1

2

3

4

5

6

7

8

9

A

pool 2 µL each

SAG192203

Tube 127

2ul SAG_S30P2R

PLT2: G6

4ul SAG_S30P2R

PLT2: G6

8ul SAG_S30P2R

PLT2: G6

mock community

2ul SAG192199

Tube 123

4ul SAG192199

Tube 123

8ul SAG192199

Tube 123

blank with ISD only

B

pool 2 µL each

SAG191508

Tube 73

2ul SAG_S38P1R

PLT2: B9

4ul SAG_S38P1R

PLT2: B9

8ul SAG_S38P1R

PLT2: B9

mock community

2ul SAG192077

Tube 96

4ul SAG192077

Tube 96

8ul SAG192077

Tube 96

blank with ISD only

C

pool 2 µL each

SAG_S16P3R

PLT1: G3

2ul SAG_S15P4R

PLT2: B2

4ul SAG_S15P4R

PLT2: B2

8ul SAG_S15P4R

PLT2: B2

mock community

2ul SAG190271

Tube 258

4ul SAG190271

Tube 258

8ul SAG190271

Tube 258

blank with ISD only

D

pool 2 µL each

SAG_S44P2R

PLT1: F7

2ul SAG190005

Tube 33

4ul SAG190005

Tube 33

8ul SAG190005

Tube 33

mock community

2ul SAG190238

Tube 255

4ul SAG190238

Tube 255

8ul SAG190238

Tube 255

blank with ISD only

E

pool according to yield

SAG192203

Tube 127

2ul SAG_S30P2R

PLT2: G6

4ul SAG_S30P2R

PLT2: G6

8ul SAG_S30P2R

PLT2: G6

mock community

2ul SAG192199

Tube 123

4ul SAG192199

Tube 123

8ul SAG192199

Tube 123

blank with ISD only

F

pool according to yield

SAG191508

Tube 73

2ul SAG_S38P1R

PLT2: B9

4ul SAG_S38P1R

PLT2: B9

8ul SAG_S38P1R

PLT2: B9

mock community

2ul SAG192077

Tube 96

4ul SAG192077

Tube 96

8ul SAG192077

Tube 96

blank with ISD only

G

pool according to yield

SAG_S16P3R

PLT1: G3

2ul SAG_S15P4R

PLT2: B2

4ul SAG_S15P4R

PLT2: B2

8ul SAG_S15P4R

PLT2: B2

mock community

2ul SAG190271

Tube 258

4ul SAG190271

Tube 258

8ul SAG190271

Tube 258

blank with ISD only

H

pool according to yield

SAG_S44P2R

PLT1: F7

2ul SAG190005

Tube 33

4ul SAG190005

Tube 33

8ul SAG190005

Tube 33

mock community

2ul SAG190238

Tube 255

4ul SAG190238

Tube 255

8ul SAG190238

Tube 255

blank with ISD only

Add 2 ul 1-step primers:
Seal with bubble seals. Vortex briefly. Spin down.
Run on Thermocycler Program GSAF36:

Temp C	Cycles	Time
95*	1X	3:00*
98	36X	0:30
62	36X	0:30
72	36X	0:30
72	1X	5:00
4	1X	0:00

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

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 all products:

Make 1:1000 dilutions of all samples 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	11	12
A	`SAG192203` Tube 127	`2ul SAG_S30P2R` PLT2: G6	`4ul SAG_S30P2R` PLT2: G6	`8ul SAG_S30P2R` PLT2: G6	mock community	`2ul SAG192199` Tube 123	`4ul SAG192199` Tube 123	`8ul SAG192199` Tube 123	blank with ISD only		NTC
B	`SAG191508` Tube 73	`2ul SAG_S38P1R` PLT2: B9	`4ul SAG_S38P1R` PLT2: B9	`8ul SAG_S38P1R` PLT2: B9	mock community	`2ul SAG192077` Tube 96	`4ul SAG192077` Tube 96	`8ul SAG192077` Tube 96	blank with ISD only	04.0002 pM Std	0.0002 pM Std
C	`SAG_S16P3R` PLT1: G3	`2ul SAG_S15P4R` PLT2: B2	`4ul SAG_S15P4R` PLT2: B2	`8ul SAG_S15P4R` PLT2: B2	mock community	`2ul SAG190271` Tube 258	`4ul SAG190271` Tube 258	`8ul SAG190271` Tube 258	blank with ISD only	0.002 pM Std	0.002 pM Std
D	`SAG_S44P2R` PLT1: F7	`2ul SAG190005` Tube 33	`4ul SAG190005` Tube 33	`8ul SAG190005` Tube 33	mock community	`2ul SAG190238` Tube 255	`4ul SAG190238` Tube 255	`8ul SAG190238` Tube 255	blank with ISD only	0.02 pM Std	0.02 pM Std
E	`SAG192203` Tube 127	`2ul SAG_S30P2R` PLT2: G6	`4ul SAG_S30P2R` PLT2: G6	`8ul SAG_S30P2R` PLT2: G6	mock community	`2ul SAG192199` Tube 123	`4ul SAG192199` Tube 123	`8ul SAG192199` Tube 123	blank with ISD only	0.2 pM Std	0.2 pM Std
F	`SAG191508` Tube 73	`2ul SAG_S38P1R` PLT2: B9	`4ul SAG_S38P1R` PLT2: B9	`8ul SAG_S38P1R` PLT2: B9	mock community	`2ul SAG192077` Tube 96	`4ul SAG192077` Tube 96	`8ul SAG192077` Tube 96	blank with ISD only	2 pM Std	2 pM Std
G	`SAG_S16P3R` PLT1: G3	`2ul SAG_S15P4R` PLT2: B2	`4ul SAG_S15P4R` PLT2: B2	`8ul SAG_S15P4R` PLT2: B2	mock community	`2ul SAG190271` Tube 258	`4ul SAG190271` Tube 258	`8ul SAG190271` Tube 258	blank with ISD only	20 pM Std	20 pM Std
H	`SAG_S44P2R` PLT1: F7	`2ul SAG190005` Tube 33	`4ul SAG190005` Tube 33	`8ul SAG190005` Tube 33	mock community	`2ul SAG190238` Tube 255	`4ul SAG190238` Tube 255	`8ul SAG190238` Tube 255	blank with ISD only

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

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	100	1000
2 ul	Primer Premix (10X)	100	200
4 ul	Ultra Pure Water	100	400
16 ul	Total Volume	100	1600

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

	1	2	3	4	5	6	7	8	9	10	11	12
A	`SAG192203` Tube 127	`2ul SAG_S30P2R` PLT2: G6	`4ul SAG_S30P2R` PLT2: G6	`8ul SAG_S30P2R` PLT2: G6	mock community	`2ul SAG192199` Tube 123	`4ul SAG192199` Tube 123	`8ul SAG192199` Tube 123	blank with ISD only	NTC	NTC	NTC
B	`SAG191508` Tube 73	`2ul SAG_S38P1R` PLT2: B9	`4ul SAG_S38P1R` PLT2: B9	`8ul SAG_S38P1R` PLT2: B9	mock community	`2ul SAG192077` Tube 96	`4ul SAG192077` Tube 96	`8ul SAG192077` Tube 96	blank with ISD only	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	`SAG_S16P3R` PLT1: G3	`2ul SAG_S15P4R` PLT2: B2	`4ul SAG_S15P4R` PLT2: B2	`8ul SAG_S15P4R` PLT2: B2	mock community	`2ul SAG190271` Tube 258	`4ul SAG190271` Tube 258	`8ul SAG190271` Tube 258	blank with ISD only	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	`SAG_S44P2R` PLT1: F7	`2ul SAG190005` Tube 33	`4ul SAG190005` Tube 33	`8ul SAG190005` Tube 33	mock community	`2ul SAG190238` Tube 255	`4ul SAG190238` Tube 255	`8ul SAG190238` Tube 255	blank with ISD only	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	`SAG192203` Tube 127	`2ul SAG_S30P2R` PLT2: G6	`4ul SAG_S30P2R` PLT2: G6	`8ul SAG_S30P2R` PLT2: G6	mock community	`2ul SAG192199` Tube 123	`4ul SAG192199` Tube 123	`8ul SAG192199` Tube 123	blank with ISD only	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	`SAG191508` Tube 73	`2ul SAG_S38P1R` PLT2: B9	`4ul SAG_S38P1R` PLT2: B9	`8ul SAG_S38P1R` PLT2: B9	mock community	`2ul SAG192077` Tube 96	`4ul SAG192077` Tube 96	`8ul SAG192077` Tube 96	blank with ISD only	2 pM Std	2 pM Std	2 pM Std
G	`SAG_S16P3R` PLT1: G3	`2ul SAG_S15P4R` PLT2: B2	`4ul SAG_S15P4R` PLT2: B2	`8ul SAG_S15P4R` PLT2: B2	mock community	`2ul SAG190271` Tube 258	`4ul SAG190271` Tube 258	`8ul SAG190271` Tube 258	blank with ISD only	20 pM Std	20 pM Std	20 pM Std
H	`SAG_S44P2R` PLT1: F7	`2ul SAG190005` Tube 33	`4ul SAG190005` Tube 33	`8ul SAG190005` Tube 33	mock community	`2ul SAG190238` Tube 255	`4ul SAG190238` Tube 255	`8ul SAG190238` Tube 255	blank with ISD only

Results:

Pool all samples:

Blue samples (top half of plate) will be pooled at 1* ul per sample.

Red samples (bottom half) will either just be pooled per qPCR numbers or concentrated via SpeedVac, reconstituted to a higher concentration, and pooled by qPCR results. (~~Pending method instructions~~).

“ul for pooling” was calculated by multiplying the lowest molarity (3.15) times the maximum V available (35 ul) to get ~110. This result was then divided by each sample’s molarity to obtain the ul needed for equimolar pooling rounded to one decimal place.

This will need to be done by hand, but if we pursue this I will tweak the Nimbus normalization program to do this. For a NovaSeq run, I would pick a higher minimum molarity. While this pool should be above the NovaSeq minimum molarity needed of 1 nM, being higher than its 3.15 nM minimal constituent, I would prefer a larger margin of safety like 5 nM. For an iSeq run, we dilute down to 50 pM, so it is not an issue.

Well Position	Sample	qPCR result	ul for pooling
1E	SAG192203_R	54.32	2.0
2E	SAG_S30P2R_R_2uL	54.14	2.0
3E	SAG_S30P2R_R_4uL	54.03	2.0
4E	SAG_S30P2R_R_8uL	11.34	9.7
5E	Mock_Comm_R	65.14	1.7
6E	SAG192199_R_2uL	71.08	1.5
7E	SAG192199_R_4uL	95.41	1.2
8E	SAG192199_R_8uL	80.56	1.4
9E	Blank_ISD_R	19.23	5.7
1F	SAG191508_R	64.52	1.7
2F	SAG_S38P1R_R_2uL	60.01	1.8
3F	SAG_S38P1R_R_4uL	56.04	2.0
4F	SAG_S38P1R_R_8uL	3.15	34.9
5F	Mock_Comm_R	63.52	1.7
6F	SAG192077_R_2uL	65.88	1.7
7F	SAG192077_R_4uL	84.27	1.3
8F	SAG192077_R_8uL	83.59	1.3
9F	Blank_ISD_R	13.12	8.4
1G	SAG_S16P3R_R	26.49	4.2
2G	SAG_S15P4R_R_2uL	53.51	2.1
3G	SAG_S15P4R_R_4uL	26.2	4.2
4G	SAG_S15P4R_R_8uL	12.91	8.5
5G	Mock_Comm_R	79.7	1.4
6G	SAG190271_R_2uL	70.2	1.6
7G	SAG190271_R_4uL	62.73	1.8
8G	SAG190271_R_8uL	59.32	1.9
9G	Blank_ISD_R	20.04	5.5
1H	SAG_S44P2R_R	19.92	5.5
2H	SAG190005_R_2uL	54.03	2.0
3H	SAG190005_R_4uL	57.05	1.9
4H	SAG190005_R_8uL	60.28	1.8
5H	Mock_Comm_R	64.83	1.7
6H	SAG190238_R_2uL	68.19	1.6
7H	SAG190238_R_4uL	55.07	2.0
8H	SAG190238_R_8uL	41.35	2.7
9H	Blank_ISD_R	18.32	6.0

qPCR blue/red pools:

Make 1:1000 dilutions of blue and red pools by adding 1 ul to 999 ul TE in a 1.5mL tubes.
Run each pool in triplicate.
Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	30	300
2 ul	Primer Premix (10X)	30	60
4 ul	Ultra Pure Water	30	120
16 ul	Total Volume	30	480

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

	1	10	11	12
A	LR2_Blue_Pool	NTC	NTC	NTC
B	LR2_Blue_Pool	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	LR2_Blue_Pool	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	LR2_Red_Pool	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	LR2_Red_Pool	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	LR2_Red_Pool	2 pM Std	2 pM Std	2 pM Std
G		20 pM Std	20 pM Std	20 pM Std
H

Results:

Average Results:

LR2_Blue_Pool: 53.65 nanomoles

LR2_Red_Pool: 35.33 nanomoles

iSeq Run

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.

Blue Pool

1000/Results = ul of Pool to Add

1000/53.7 = 18.6 uL of Pool to Add

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

1000- 18.6 = 981.4 uL of 10mM Tris 8.5

Red Pool

1000/Results = ul of Pool to Add

1000/35.3 = 28.3 uL of Pool to Add

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

1000- 28.3= 971.7 uL of 10mM Tris 8.5

Combine Blue and Red pools in equal parts once pools are at 1nM

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.
Results located: Data/SequencingRuns/”folder with applicable date”/Alignment_1/Fastq/*.fastq.gz
Upload data through Globus portal off external drive

Data wrangling(2):

Use R to associate NS2 input concentrations with sample names, read counts, and % ISD reads

	1	2	3	5	6	7	9
A	6ul	6ul	4ul	6ul	6ul	4ul	6ul
B	6ul	6ul	4ul	6ul	6ul	4ul	6ul
C	6ul	6ul	4ul	6ul	6ul	4ul	6ul
D	6ul	6ul	4ul	6ul	6ul	4ul	6ul
E	6ul	6ul	4ul	6ul	6ul	4ul	6ul
F	6ul	6ul	4ul	6ul	6ul	4ul	6ul
G	6ul	6ul	4ul	6ul	6ul	4ul	6ul
H	6ul	6ul	4ul	6ul	6ul	4ul	6ul

	1	2	3	5	6	7	9
A	6ul	6ul	4ul	6ul	6ul	4ul	6ul
B	6ul	6ul	4ul	6ul	6ul	4ul	6ul
C	6ul	6ul	4ul	6ul	6ul	4ul	6ul
D	6ul	6ul	4ul	6ul	6ul	4ul	6ul
E	6ul	6ul	4ul	6ul	6ul	4ul	6ul
F	6ul	6ul	4ul	6ul	6ul	4ul	6ul
G	6ul	6ul	4ul	6ul	6ul	4ul	6ul
H	6ul	6ul	4ul	6ul	6ul	4ul	6ul

	1	2	3	5	6	7	9
A	6ul	6ul	4ul	6ul	6ul	4ul	6ul
B	6ul	6ul	4ul	6ul	6ul	4ul	6ul
C	6ul	6ul	4ul	6ul	6ul	4ul	6ul
D	6ul	6ul	4ul	6ul	6ul	4ul	6ul
E	6ul	6ul	4ul	6ul	6ul	4ul	6ul
F	6ul	6ul	4ul	6ul	6ul	4ul	6ul
G	6ul	6ul	4ul	6ul	6ul	4ul	6ul
H	6ul	6ul	4ul	6ul	6ul	4ul	6ul