Project: Thermal Transplant Molecular

Goal

First trial of WGBS library prep using the Zymo Pico Methyl-seq library prep kit and this protocol.

Sample dilution and mix calculations can be found on this google sheet

Protocol

I am using a modified version of this protocol.

The only modification I made to this protocol was changing the input DNA from 1ng to 10ng. This worked in previous library preps performed by E. Strand and M. Schedl.

Samples and Indices

Sample	Coral.ID	Life Stage	i5 index #	i7 index #
18-358	P-10-A	ADULT	1	1
18-20	R-7-B	ADULT	2	2
L-933	P-10-A	LARVAE	3	3

Qubit Results

To test DNA quantity: Qubit

I used the DNA broad range assay.

Sample	DNA 1 (ng/ul)	DNA 2 (ng/ul)
Standard 1	184.99
Standard 2	19664.63
18-358 (#1)	21.6	21.0
18-20 (#2)	12.2	11.8
L-933 (#3)	5.80	5.64

Tapestation Results

D5000 Tapestation protocol

Full Tapestation Report

KW #1 (18-358):

KW #2 (18-20):

KW #3 (L-933):

Summary

Overall, this protocol worked, however there is PCR over-amplification. We will need to optimize the number number of PCR cycles to reduce this over-amplification.