Project: Thermal Transplant Molecular

Goal

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

Today, I wanted to test 2 different modifications during the second amplification:

Modification #1:

• Use the combined i5 and i7 index primers (an input of 1uL)
• Reduce the Library Amp Master Mix (2x) to 13uL (was originally 14uL)

Modification #2:

• Use the KAPA HiFi HotStart ReadyMix (2X) instead of the Library Amp Master Mix (2x)

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

Protocol

I am using a modified version of this protocol.

Samples and Indices

Sample	Coral ID	Life Stage	i5 Index #	i7 Index #	Modification #
18-118	P-6-A	Adult	7	7	1
18-106	P-19-A	Adult	8	8	1
18-334	P-19-B	Adult	9	9	2
18-442	R-9-B	Adult	10	10	2
18-167	R-19-A	Adult	11	11	NA
L-1053	P-12-A	Larvae	12	12	NA
L-1093	R-19-A	Larvae	13	13	NA
L-704	P-15-A	Larvae	14	14	NA

Qubit Results

To test DNA quantity: Qubit

I used the DNA broad range assay.

I ran the qubit and tapestation on the following day (20210423). After the bead clean up step I stored the libraries in the fridge at 4C for ~16 hours.

Sample	DNA 1 (ng/ul)	DNA 2 (ng/ul)
Standard 1	176.98
Standard 2	19057.22
18-118 (#7)	25.2	24.8
18-106 (#8)	25.2	24.8
18-334 (#9)	28.0	27.8
18-442 (#10)	73.2	72.0
18-167 (#11)	29.4	28.8
L-1053 (#12)	8.90	8.50
L-1093 (#13)	24.6	24.0
L-704 (#14)	7.08	6.92

Tapestation Results

D5000 Tapestation protocol

Full Tapestation Report

Summary

Both modifications produced good libraries. The KAPA HiFi modification increased the yield. This also occurred in previous library preps.