WGBS Pico Methylation Protocol Trials

Putnam Lab WGBS Protocol

Google spreadsheet with all WGBS raw data from E.Strand

As 20201112, two test runs have been done for WGBS PMS protocol to optimize sample input for five species of coral: P. lutea, M. capitata, P. acuta, P. meandrina, P. asterodies.

Both trials done in 2 days - day 1 ends with the bisulfite conversion step, left in the 4C fridge for less than 20 hours and then started again the next day. This could probably be done in 1 day in the future now that I have done this protocol multiple times.

Trial 1: 20201015 and 20201026

DNA input: 1 ng. Dilutions done by taking 2 uL of DNA + (1-Qubit value)x2 10 uM Tris HCl.

BR DNA Standard 1: 200.79
BR DNA Standard 2: 22,255.60

Coral_ID	Species	Qubit (ng/uL)	Gel Pass?	DNA for dilution (ul)	10 mM Tris for dilution (ul)	Qubit (ng/uL) post PMS	TapeStation pass?	Notes
P7	Pocillopora meandrina	75.6	Not good	2	149.2	Too low	Peaks visible	Hawaii - Eva extracted this with HMW kit and sent it in the recent dry shipper
P3	Porites lutea	136.5	Not good	2	271	Too low	Peaks visible	Hawaii - Eva extracted this with HMW kit and sent it in the recent dry shipper
P1	Porites astreoides	8.27	Great	2	14.54	2.08	Peaks visible	genome coral - Maggie extracted this with HMW kit

Date	Sample	i5 index #	i7 index #
20201025	P1 - Past	1	1
20201025	P3 - P lutea	2	2
20201025	P7 - Poc mea	3	3

Calculations for mixes:

Amplification with Prep-Amp Primers
3 samples + 0.15 (for 5% error) = 3.15

Priming Master Mix (PMM):
6.3 uL of 5X PrepAmp Buffer (2 uL * 3.15 samples = 6.3)
3.15 uL 40 uM PrepAmp Primer (1 uL * 3.15 samples = 3.15)

PrepAmp Master Mix (PAMM):
3.15 uL of 5X PrepAmp Buffer (1 uL * 3.15 samples = 3.15)
11.81 uL PrepAmp Pre Mix (3.75 uL * 3.15 samples = 11.81)
0.95 uL PrepAmp Polymerase (0.3 uL * 3.15 = 0.95)

“Diluted” PrepAmp Polymerase mix
0.9 uL PrepAmp Polymerase (0.3 uL * 3 samples = 0.9)
6.3 uL DNA Elution Buffer (0.2 uL * 3.15 = 6.3)
*The above calculation should be 0.63 uL and 0.99 uL, unclear if that was a written mistake or a mistake made during the protocol. This mix could have been far too diluted for the protocol, leading to less enzymatic activity and smaller output.**

1st Amplification Master Mix (AMM)
39.38 uL Library Amp Mix (2X) (12.5 uL * 3.15 samples = 39.375)
3.15 uL Library Amp Primers (10 uM) (1 uL * 3.15 samples = 3.15)

20201026 Trial 1 TapeStation Report

Trial 2: 20201110 and 20201111

DNA input: 10 ng. 10 ng/uL calculations done by 100/Qubit value uL DNA for dilution and 10-DNA value for 10 uM Tris HCl volume.

BR DNA Standard 1: 180.28
BR DNA Standard 2: 19,255.88

Coral_ID	Species	Qubit (ng/uL)	Gel Pass?	DNA needed (uL)	10 mM Tris HCl (uL)	Qubit (ng/uL) post PMS	TapeStation pass?	Notes
P3	Pocillopora meandrina	54.75	Good	1.83	8.17	3.81	Peaks visible	Maggie extracted HMW from 20201109
P1	Porites astreoides	8.27	Good	Too low to dilute; just take 1 uL of original tube		Too low	Peaks visible	genome coral - Maggie extracted this with HMW kit
2860	Montipora capitata	43.5	Good	2.30	7.70	2.58	Peaks visible	Emma extracted - Holo Int
2878	Pocillopora acuta	48.1	Fair	2.08	7.92	3.81	Peaks visible	Emma extracted - Holo Int

Index Key

Sample Name/Pool Name*	Library Name*	i7 Index Name*	i7 Index Sequence*	i5 Index Name*	i5 Index Sequence*
Mcap_2860	Mcap_2860	i7_UDI0001	AACCGCGG	i5_UDI0001	AGCGCTAG
Pact_2878	Pact_2878	i7_UDI0002	GGTTATAA	i5_UDI0002	GATATCGA
Past_P1	Past_P1	i7_UDI0003	CCAAGTCC	i5_UDI0003	CGCAGACG
Pmean_P3	Pmean_P3	i7_UDI0004	TTGGACTT	i5_UDI0004	TATGAGTA

I started my samples at 1 and 2 in case this trial worked and I could start the rest of my samples at 3.

Mix Calculations

Amplification with Prep-Amp Primers
4 samples + 0.2 (for 5% error) = 4.2

Priming Master Mix (PMM):
8.4 uL of 5X PrepAmp Buffer (2 uL * 4.2 samples = 8.4)
4.2 uL 40 uM PrepAmp Primer (1 uL * 4.2 samples = 4.2)

PrepAmp Master Mix (PAMM):
4.2 uL of 5X PrepAmp Buffer (1 uL * 4.2 samples = 4.2)
15.75 uL PrepAmp Pre Mix (3.75 uL * 4.2 samples = 15.75)
1.26 uL PrepAmp Polymerase (0.3 uL * 4.2 = 1.26)

“Diluted” PrepAmp Polymerase mix
1.26 uL PrepAmp Polymerase (0.3 uL * 4.2 samples = 1.26)
0.84 uL DNA Elution Buffer (0.2 uL * 4.2 = 0.84)

1st Amplification Master Mix (AMM)
52.5 uL Library Amp Mix (2X) (12.5 uL * 4.2 samples = 52.5)
4.2 uL Library Amp Primers (10 uM) (1 uL * 4.2 samples = 4.2)

20201111 Trial 2 TapeStation Report

The concentrations are much higher on the TapeStation than Qubit values. Which one do we trust more? The output of WGBS PMS is only 15 uL max, and I used 3 uL for tapestation and qubit (prepped qubit twice). I’m hesitant to use more to re-qubit unless we have to.

Submission details (20201117_30-445814566_Strand_WGBS):

Sample #	General Information						Amount of Material			Miscellaneous		Special Comments
	SeqFile-Name	Sample/Pool Name*	Number of Libraries in Pool*	Starting Material Type*	Species/Strain*	Sample Buffer*	Total Amount*	Volume (µL)*	Conc. (nM or ng/uL)*	Purification Method	Library Prep Kit Used*
1	ES1	Mcap_2860	1	DNA	Porites astreoides	DNA elution buffer	25.8	10	2.58	KAPA Pure Beads	Zymo Pico Methyl-Seq Library Prep Kit
2	ES2	Pact_2878	1	DNA	Pocillopora meandrina	DNA elution buffer	38.1	10	3.81	KAPA Pure Beads	Zymo Pico Methyl-Seq Library Prep Kit
3	ES3	Past_P1	1	DNA	Montipora capitata	DNA elution buffer	10	10	1	KAPA Pure Beads	Zymo Pico Methyl-Seq Library Prep Kit
4	ES4	Pmean_P3	1	DNA	Pocillopora acuta	DNA elution buffer	38.1	10	3.81	KAPA Pure Beads	Zymo Pico Methyl-Seq Library Prep Kit