Project: Thermal Transplant Molecular, Holobiont Integration (ES)

Goal

Re-amplify PicoMethyl-seq libraries that had low output (<5ng/ul) from initial prep using this protocol. I am also re-amplifying samples for E. Strand that had low output from the PicoMethyl-seq prep.

Protocol

Repeating the last two steps of this protocol:

1) Second amplification with 4 cycles instead of 10 cycles

2) 1X bead clean up

For all samples, I used the following inputs for the second amplification:

• 12 uL of the PicoMethyl-seq library
• 13 uL of Library Amp Master Mix (2x)
• 1 uL of the combined i5 and i7 index primers (I used the same indices as the original library prep)

Samples, indices, original Qubit quantification, post re-amplification Qubit quantification

KW/ES	Sample #	Vial	i5/i7 index #	Original Library (ng/uL)	DNA 1 (ng/uL)	DNA 2 (ng/uL)
KW	49	18-358	49	5.96	20.4	20.4
KW	52	L-924	52	2.50	26.6	26.4
ES	7	1707	7	2.78	12.7	12.6
ES	8	2212	8	5.94	25.0	24.8
ES	10	2861	10	LOW	LOW	LOW
ES	15	1103	15	LOW	LOW	LOW
	Qubit std.1				178.04
	Qubit std.2				19128.66

Tapestation Results

D5000 Tapestation protocol

Full Tapestation Report