Using the Zymo Pico Methyl Seq Library Prep Kit for the 2nd Set of 16 of Danielle’s Pocillopora DNA Samples and Sample E7 Again for Whole Genome Bisulfite Sequencing
Goal Library prep 17 samples for WGBS
Results 15 samples were prepped successfully, 2 did not work
Takeaways E7 worked this time, I am not sure why E2 and E9 did not. I will have to prep them again. Also the KAPA HiFi HotStart Ready Mix can be substituted in the last amplification, but it might over amplify them a little bit? Those libraries amplified way better than the others. Increasing the number of cycles to 13 didn’t seem to do anything to help library yield.
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 01 Bisulfite Conversion
- Samples used for second prep:
- E7
- E6
- C21
- E8
- E12
- E5
- C30
- E1
- C23
- E16
- C19
- C25
- E2
- E9
- C32
- E15
- C17
- Followed exact steps as in protocol
- Once the thermocycler program was done the samples were put in the 4 degree fridge overnight
2020 10 02
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 * 17.7 = 35.4ul
- 1ul PrepAmp Primer * 17.7 = 17.7ul
- PrepAmp Master Mix calculations (PAMM):
- 1ul PrepAmp Buffer * 17.7 = 17.7ul
- 3.75ul PrepAmp PreMix * 17.7 = 66.38ul
- 0.3ul PrepAmp Polymerase * 17.7 = 5.31ul
- Dilution calculation of PrepAmp Polymerase to add 0.5ul:
- 0.3 PrepAmp Polymerase * 17.7 = 5.31ul
- 0.2ul DNA elution buffer * 17.7 = 3.54ul
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 * 17.7 = 221.25ul
- 1ul Library Amp Primers * 17.7 = 17.7ul
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
- Thermocycler program had 13 cycles this time
- For the last two samples, E15 and C17, there was not enough Library Amp Mix in the kit reagent box, so I used the same volume of KAPA HiFi HotStart Ready Mix, another amplification mix, and hoped it would work
- Table for components in tubes for amplifications:
| Sample |
Volume DNA (ul) |
Volume Library Amp Mix (ul) |
Volume i5 Primer (10uM) |
Volume i7 Primer (10uM) |
| E7 |
12 |
14 |
1ul i5_ZM_UDI002 |
1ul i7_ZM_UDI002 |
| E6 |
12 |
14 |
1ul i5_ZM_UDI017 |
1ul i7_ZM_UDI017 |
| C21 |
12 |
14 |
1ul i5_ZM_UDI018 |
1ul i7_ZM_UDI018 |
| E8 |
12 |
14 |
1ul i5_ZM_UDI019 |
1ul i7_ZM_UDI019 |
| E12 |
12 |
14 |
1ul i5_ZM_UDI020 |
1ul i7_ZM_UDI020 |
| E5 |
12 |
14 |
1ul i5_ZM_UDI0021 |
1ul i7_ZM_UDI021 |
| C30 |
12 |
14 |
1ul i5_ZM_UDI022 |
1ul i7_ZM_UDI022 |
| E1 |
12 |
14 |
1ul i5_ZM_UDI023 |
1ul i7_ZM_UDI023 |
| C23 |
12 |
14 |
1ul i5_ZM_UDI024 |
1ul i7_ZM_UDI024 |
| E16 |
12 |
14 |
1ul i5_ZM_UDI025 |
1ul i7_ZM_UDI025 |
| C19 |
12 |
14 |
1ul i5_ZM_UDI026 |
1ul i7_ZM_UDI026 |
| C25 |
12 |
14 |
1ul i5_ZM_UDI027 |
1ul i7_ZM_UDI027 |
| E2 |
12 |
14 |
1ul i5_ZM_UDI028 |
1ul i7_ZM_UDI028 |
| E9 |
12 |
14 |
1ul i5_ZM_UDI029 |
1ul i7_ZM_UDI029 |
| C32 |
12 |
14 |
1ul i5_ZM_UDI030 |
1ul i7_ZM_UDI030 |
| E15 |
12 |
14 KAPA mix |
1ul i5_ZM_UDI031 |
1ul i7_ZM_UDI031 |
| C17 |
12 |
14 KAPA mix |
1ul i5_ZM_UDI032 |
1ul i7_ZM_UDI032 |
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
| Sample |
Reading 1 (ng/ul) |
Reading 2(ng/ul) |
Average (ng/ul) |
| Standard 1 |
182 RFU |
- |
- |
| Standard 2 |
19839 RFU |
- |
- |
| E7 |
5.76 |
5.62 |
5.69 |
| E6 |
18.4 |
18.3 |
18.35 |
| C21 |
18.9 |
18.7 |
18.8 |
| E8 |
12.9 |
12.9 |
12.9 |
| E12 |
8.46 |
8.4 |
8.43 |
| E5 |
19.2 |
19.3 |
19.25 |
| C30 |
17.8 |
18 |
17.9 |
| E1 |
18.4 |
18.3 |
18.35 |
| C23 |
20.2 |
20.2 |
20.2 |
| E16 |
19.3 |
19.3 |
19.3 |
| C19 |
19 |
18.9 |
18.95 |
| C25 |
22.4 |
22.2 |
22.3 |
| E2 |
too low |
- |
- |
| E9 |
too low |
- |
- |
| C32 |
14.6 |
14.4 |
14.5 |
| E15 |
42.8 |
42.8 |
42.8 |
| C17 |
55.4 |
55 |
55.2 |
2020 10 05 D5000 TapeStation
Samples and Index Sequences
| Coral ID |
i7 bases |
i5 bases |
| E7 |
TTATAACC |
GATATCGA |
| E6 |
TAATACAG |
ATATTCAC |
| C21 |
CGGCGTGA |
GCGCCTGT |
| E8 |
ATGTAAGT |
ACTCTATG |
| E12 |
GCACGGAC |
GTCTCGCA |
| E5 |
GGTACCTT |
AAGACGTC |
| C30 |
AACGTTCC |
GGAGTACT |
| E1 |
GCAGAATT |
ACCGGCCA |
| C23 |
ATGAGGCC |
GTTAATTG |
| E16 |
ACTAAGAT |
AACCGCGG |
| C19 |
GTCGGAGC |
GGTTATAA |
| C25 |
CTTGGTAT |
CCAAGTCC |
| C32 |
TTACAGGA |
TGACAAGC |
| E15 |
GGCATTCT |
CTAGCTTG |
| C17 |
AATGCCTC |
TCGATCCA |
Representative library trace:
E8 Library that is smaller than all the others:
