Using the Zymo Pico Methyl Seq Kit on the MBD enriched Eastern Oyster DNA from samples from Rebecca Stevick

Dilution of MBD enriched DNA samples 2020-01-08

While we haven’t tested this kit on oyster DNA, my plan was to start with 1ng input DNA into the kit like what has worked for coral DNA. First the samples had to be diluted 1:2 to avoid pipetting less than a µl of DNA

Sample	volume DNA	volume ultra pure water
1	5	10
2	5	10
3	5	10
4	5	10
5	5	10
6	5	10
7	5	10
8	5	10
9	5	10
10	5	10
11	5	10
12	5	10

Sample Prep for PMS

Samples need to all include 1ng of sample, .05ng spike-in, and water up to 20µl

sample	volume DNA	volume diluted spike	volume ultra pure water to 20µl
1	1.3µl	2µl	16.7µl
2	1.2µl	2µl	16.8µl
3	1.7µl	2µl	16.3µl
4	1.3µl	2µl	16.7µl
5	1.7µl	2µl	16.3µl
6	1.3µl	2µl	16.7µl
7	2.1µl	2µl	15.9µl
8	3.6µl	2µl	14.4µl
9	1.5µl	2µl	16.5µl
10	1.7µl	2µl	16.3µl
11	2.2µl	2µl	15.8µl
12	1.5µl	2µl	16.5µl

Bisulfite Conversion

• Added 130µl lightning conversion reagent to each sample in at PCR tube
• Put tubes in the thermocycler Pico bisulfite conversion program
• After program sample tubes were put in the 4 degree fridge overnight

Next Day 2020-01-09 Cleanup

• Made 12 spin column, one for each sample
• Added 600µl M-binding buffer each to 12 spin columns
• Added 150µl of the BS reaction (all) to each individual tube
• Invert columns to mix
• Centrifuged columns at 12,000 rcf for 30 seconds and discarded flowthrough
• Added 100µl M-Wash buffer to each column
• Centrifuged columns at 12,000 rcf for 30 seconds and discarded flowthrough
• Added 200µl L-desulfonation buffer to each column
• Let them sit for 15 minutes
• Centrifuged columns at 12,000 rcf for 30 seconds and discarded flowthrough
• Added 200µl M-wash buffer to each column
• Centrifuged columns at 12,000 rcf for 30 seconds and discarded flowthrough
• Added 200µl M-wash buffer to each column
• Centrifuged columns at 12,000 rcf for 2 minutes and discarded flowthrough
• Added 8µl warmed (56C) DNA elution buffer to each column and let sit for 5 minutes in new 1.5mL tubes to collect
• Centrifuged columns at 12,000 rcf for 30 seconds

Amplification with PrepAmp Primers

• Made Priming master mix on ice:
  • 2µl 5X PrepAmp buffer * 12.5 = 25µl
  • 1µl PrepAmp Primers (40µM) * 12.5 = 12.5µl
• Made new PCR tubes with 3µl of Priming MM and 7µl of bisulfite treated DNA
• Kept those on ice
• Made PrepAmp Mix on ice:
  • 1µl 5X PrepAmp buffer * 12.5 = 12.5µl
  • 3.75µl PrepAmp PreMix * 12.5 = 46.9µl
  • 0.3µl PrepAmp polymerase * 12.5 = 3.75µl
• Made slightly diluted PrepAmp polymerase to avoid adding less than .5µl:
  • 3.75µl PrepAmp polymerase
  • 2.5µl elution buffer
• Set thermocylcer program with lid temp restricted to 25 degrees C and place samples inside and run the pico priming program on the thermocycler:
  • 98 for 2 minutes
  • 8 degrees for 1 minute
  • 8 degree hold
  • During hold vortex, spin tubes down, add 5.05µl PrepAmp Mix to each tube, vortex, spin down, and place back in thermocycler
  • 8 degrees for 4 minutes
  • 16 degrees for 1 minute with 3% ramp rate
  • 22 degrees for 1 minute with 3% ramp rate
  • 28 degrees for 1 minute with 3% ramp rate
  • 36 degrees for 1 minute with 3% ramp rate
  • 36.5 degrees for 1 minute with 3% ramp rate
  • 37 degrees for 8 minutes
  • repeat back from the first step one time through again
  • During hold, vortex, spin tubes down, tried to add 0.3µl of slightly diluted PrepAmp polymerase, vortex, spin down, and place back into thermocycler

Cleanup with DNA Clean and Concentrator Columns (DCC)

• Made a 1.5mL tube for each sample, added 7:1 ratio DNA binding buffer, so 107.45µl of DNA binding buffer
• Put elution buffer in thermomixer 56 degrees
• Added DNA sample (15.35µl) to the appropriate 1.5mL tube
• Vortexed, spun down, and added to the column
• Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
• Added 200µl M-wash buffer to each column
• Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
• Added 200µl M-wash buffer to each column
• Centrifuged 12,000 rcf for 2 minutes, discarded flowthrough
• Transferred columns to 1.5mL tubes
• Added 12µl warmed elution buffer to each column directly
• Incubated 5 minutes
• Centrifuged 12,000 rcf 30 seconds

First Amplification

• Made 1st Amp master mix:
  • 12.5µl 2X Library Amp Mix * 12.5 = 156.25µl
    • 1µl Library Amp Primer(10µM) * 12.5 = 12.5µl
• Added 13.5µl MM to new PCR tubes
• Added 11.5µl of cleaned and concentrated DNA sample to the appropriate new PCR tube
• Vortexed, spun down, and placed in thermocycler program 1st Pico Methyl Amp program 8 cycles for the 1ng input

Cleanup with DCC

• Made a 1.5mL tube for each sample, added 7:1 ratio DNA binding buffer, so 175µl of DNA binding buffer
• Put elution buffer in thermomixer 56 degrees
• Added DNA sample (25µl) to the appropriate 1.5mL tube
• Vortexed, spun down, and added to the column
• Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
• Added 200µl M-wash buffer to each column
• Centrifuged 12,000 rcf 30 seconds, discarded flowthrough
• Added 200µl M-wash buffer to each column
• Centrifuged 12,000 rcf 2 minutes, discarded flowthrough
• Transferred columns to 1.5mL tubes
• Added 12.5µl warmed elution buffer to each column directly
• Incubated 5 minutes
• Centrifuged 12,000 rcf 30 seconds

Amplification with Index Primers

• In PCR tubes combined the following:

  sample	volume DNA	volume LibAmp MM	volume i5 primer	volume i7 primer
  1	12µl	14µl	1µl 1	1µl 1
  2	12µl	14µl	1µl 2	1µl 2
  3	12µl	14µl	1µl 3	1µl 3
  4	12µl	14µl	1µl 4	1µl 4
  5	12µl	14µl	1µl 5	1µl 5
  6	12µl	14µl	1µl 6	1µl 6
  7	12µl	14µl	1µl 7	1µl 7
  8	12µl	14µl	1µl 8	1µl 8
  9	12µl	14µl	1µl 9	1µl 9
  10	12µl	14µl	1µl 10	1µl 10
  11	12µl	14µl	1µl 11	1µl 11
  12	12µl	14µl	1µl 12	1µl 12

• Vortexed, spun down, and placed in thermocycler program 2nd Pico Methyl Amp program (10 cycles)

1X Bead Cleanup

• Allowed beads to come to room temp after resuspending
• Made fresh 80% EtOH
• Added equal volume of beads to each sample: 26µl
• Pipetted to mix
• Incubated on shaker 15 minutes
• Followed normal bead cleanup procedure
• Resuspended and eluted beads in 15µl elution buffer

Broad Range Qubit

Standard 1	Standard 2	Sample	Average DNA ng/µl
168 RFU	22757 RFU	1	2.01
-	-	2	2
-	-	3	3.4
-	-	4	2
-	-	5	2.01
-	-	6	2.42
-	-	7	2.34
-	-	8	2.03
-	-	9	2.12
-	-	10	4.32
-	-	11	2.48
-	-	12	2

Not great concentrations, and we are out of DNA screen tapes. But it probably worked fine and just needs to be amplified further.

4 Cycle PCR and Cleanup

• Split up libraries by two to do two amplifications for each
• Made 24 PCR tubes with 7µl of sample in each
• Made a master mix of LibraryAmp Mix and primers and water for each sample pair

sample mix	volume LibAmp MM	volume i5 primer	volume i7 primer	volume water
1	24µl	2µl 1	2µl 1	8µl
2	24µl	2µl 2	2µl 2	8µl
3	24µl	2µl 3	2µl 3	8µl
4	24µl	2µl 4	2µl 4	8µl
5	24µl	2µl 5	2µl 5	8µl
6	24µl	2µl 6	2µl 6	8µl
7	24µl	2µl 7	2µl 7	8µl
8	24µl	2µl 8	2µl 8	8µl
9	24µl	2µl 9	2µl 9	8µl
10	24µl	2µl 10	2µl 10	8µl
11	24µl	2µl 11	2µl 11	8µl
12	24µl	2µl 12	2µl 12	8µl

• Added 18µl of the appropriate master mix to each tube
• Placed in thermocycler 2nd Amp program 4 cycles

1X Bead Cleanup

• Allowed beads to come to room temp after resuspending
• Made fresh 80% EtOH
• Duplicate amplifications were pooled back together for a total of 50µl
• Added equal volume of beads to each sample: 50µl
• Pipetted to mix
• Incubated on shaker 15 minutes
• Followed normal bead cleanup procedure
• Resuspended and eluted beads in 14µl elution buffer

Broad Range Qubit

Standard 1	Standard 2	Sample	Average DNA ng/µl
154 RFU	22290 RFU	1	18
-	-	2	18.7
-	-	3	30.8
-	-	4	19
-	-	5	13
-	-	6	26.3
-	-	7	23.8
-	-	8	22.5
-	-	9	20.2
-	-	10	39.2
-	-	11	26.8
-	-	12	14

D5000 TapeStation

Size distributions of libraries, and one example trace:

Full results

Final Sample Information

Sample ID	Data_Name	Sample_Num	Sample_Name	Bucket	Treatment	Library ng/ul	i5 Index	Index i5 Sequence	i7 Index	Index i7 Sequence
1	CON7_1	89	BC.7.1	7	Control	18	1	AGCGCTAG	1	CCGCGGTT
2	CON7_2	90	BC.7.2	7	Control	18.7	2	GATATCGA	2	TTATAACC
3	CON7_3	91	BC.7.3	7	Control	30.8	3	CGCAGACG	3	GGACTTGG
4	CON9_1	95	BC.9.1	9	Control	19	4	TATGAGTA	4	AAGTCCAA
5	CON9_2	96	BC.9.2	9	Control	13	5	AGGTGCGT	5	ATCCACTG
6	CON9_3	97	BC.9.3	9	Control	26.3	6	GAACATAC	6	GCTTGTCA
7	ENR11_1	101	BC.11.1	11	Enriched	23.8	7	ACATAGCG	7	CAAGCTAG
8	ENR11_2	102	BC.11.2	11	Enriched	22.5	8	GTGCGATA	8	TGGATCGA
9	ENR11_3	103	BC.11.3	11	Enriched	20.2	9	CCAACAGA	9	AGTTCAGG
10	ENR12_1	104	BC.12.1	12	Enriched	39.2	10	TTGGTGAG	10	GACCTGAA
11	ENR12_2	105	BC.12.2	12	Enriched	26.8	11	CGCGGTTC	11	TCTCTACT
12	ENR12_3	106	BC.12.3	12	Enriched	14	12	TATAACCT	12	CTCTCGTC