Using the Zymo Pico Methyl Seq Library Prep Kit for Two Final of Danielle’s Pocillopora DNA Samples for Whole Genome Bisulfite Sequencing

Goal Library prep 2 samples for WGBS that failed previously
Results Both samples worked!
Takeaways I re-diluted the sample DNA and the index primers, so I do not know if either of those was the initial problem or something else during the prep. I continue to struggle to get all preps to work on the first prep, I am not sure why. It was noticeably easier and much more manageable to do 2 samples instead of 16/17. I think that might be almost too many to do at once.

This library prep followed the exact protocol for the Zymo Pico Methyl Seq Kit from me. See that protocol for detailed descriptions of each steps. Tables and values specific for this prep are included below.

2020 10 13

Re-Dilution of Samples:

Coral ID	DNA for dilution to 1ng/ul (ul)	10mM Tris HCl for dilution to 1ng/ul (ul)
E2	2	20.6
E9	2	20.8

Re-Dilution of Index Primers

Primer Name	Volume 200uM Stock	Volume Nuclease-free Water for 10uM
i5_ZM_UDI028	10ul	190ul
i7_ZM_UDI028	10ul	190ul
i5_ZM_UDI029	10ul	190ul
i7_ZM_UDI029	10ul	190ul

Bisulfite Conversion

• Samples used in prep:
  • E2
  • E9
• Followed exact steps as in protocol
• Proceeded directly to next steps

Post-BS Conversion cleanup

• Followed exact steps as in the protocol

Amplification with Prep-Amp Primers

• Followed exact steps as in the protocol
• Priming Master Mix calculations (PMM):
  • 2ul PrepAmp Buffer * 2.2 = 34.4ul
  • 1ul PrepAmp Primer * 2.2 = 2.2ul
• PrepAmp Master Mix calculations (PAMM):
  • 1ul PrepAmp Buffer * 2.2 = 2.2ul
  • 3.75ul PrepAmp PreMix * 2.2 = 8.25ul
  • 0.3ul PrepAmp Polymerase * 2.2 = 0.66ul
• Dilution calculation of PrepAmp Polymerase to add 0.5ul:
  • 0.3 PrepAmp Polymerase * 2.2 = 0.66ul
  • 0.2ul DNA elution buffer * 2.2 = 0.34ul
• Note: it was very hard to pipette 0.66, this may have been slightly off

DNA Clean and Concentrator

• Followed exact steps as in the protocol

First Amplification

• Followed exact steps as in the protocol
• 1st Amp Master Mix calculation:
  • 12.5ul Library Amp Mix * 2.2 = 27.5ul
  • 1ul Library Amp Primers * 2.2 = 2.2ul

Second DNA Clean and Concentrator

• Followed exact steps as in the protocol

Second Amplification with Index Primers

• Followed exact steps as in the protocol, except see below
• Table for components in tubes for amplifications:

Sample	Volume DNA (ul)	Volume Library Amp Mix (ul)	Volume i5 Primer (10uM)	Volume i7 Primer (10uM)
E2	12	14	1ul i5_ZM_UDI028	1ul i7_ZM_UDI028
E9	12	14	1ul i5_ZM_UDI029	1ul i7_ZM_UDI029

1X Bead Clean

• Followed exact steps as in protocol
• Samples were Qubited immediately so they were put on an ice bucket not frozen yet

Broad Range dsDNA Qubit

• Followed Qubit protocol

Sample	Reading 1 (ng/ul)	Reading 2(ng/ul)	Average (ng/ul)
Standard 1	181 RFU	-	-
Standard 2	19823 RFU	-	-
E2	13.8	13.7	13.75
E9	15.4	15.3	15.35

D5000 TapeStation

• Followed TapeStation protocol
• TapeStation report link E2 Library: E9 Library:

Samples and Index Sequences

Coral ID	i7 bases	i5 bases
E2	TCCAACGC	TTGGACTT
E9	CCGTGAAG	CAGTGGAT