Doing a 1 Cycle Reconditioning PCR on KBay WGBS Libraries That Looked Over-Amplified/Overly Broad Peaks

Goal Try 1 cycle PCR on some libraries with a broad or double peak to remove potential PCR bubbles, as stated here
Results All libraries have normal peaks after PCR
Takeaways Looks like this process works really well, all peaks look pretty much normal now. However, this process does use up reagents from the library prep kit that are limiting to us

10 Samples were chosen because their tapestations showed either double peaks or very broad peaks

For each sample I repeated the 2nd amplification from the WGBS protocol. The samples had more than 12ul volume in the final libraries, so if this did not work I knew I had some saved that we could use. The PCR program was copied and the program changed to have only 1 cycle. This is called 1 PICO METHYL AMP 2 in the thermocycler program directory.

Extraction-ID	Library Volume	KAPA Mix Volume	Primer Pair	Volume Primer
22	12ul	13ul	2	1ul
26	12ul	13ul	3	1ul
38	12ul	13ul	11	1ul
34	12ul	13ul	18	1ul
50	12ul	13ul	21	1ul
33	12ul	13ul	24	1ul
54	12ul	13ul	26	1ul
39	12ul	13ul	27	1ul
56	12ul	13ul	31	1ul
59	12ul	13ul	40	1ul

After the PCR (13 minutes), I performed the normal 1X bead cleanup as outlined in the protocol. The libraries were resuspended in 17ul elution buffer.

Broad Range dsDNA Qubit

• Followed Qubit protocol

Sample	Reading 1 (ng/ul)	Reading 2(ng/ul)	Average (ng/ul)
S1	178 RFU	-	-
S2	20137 RFU	-	-
22	54.4	54.2	54.3
26	53.2	53	53.1
38	54.6	54.8	54.7
34	53.4	53	53.2
50	46	45.6	45.8
33	50.4	50.2	50.3
54	47.8	48.2	48
39	51	50.8	50.9
56	37.8	37.6	37.5
59	51.6	51	51.3

D5000 TapeStation

• Followed TapeStation protocol
• I used two tapes
• Link 1
• Link 2

Example before and after traces, clearly two peaks visible in the first one:
Before

After

Example before and after traces, sample that may have two peaks or just one big one:
Before

After