2nd 12 WGBS of K-Bay Bleaching Samples

Using the Zymo Pico Methyl Seq Library Prep Kit For Another 12 Montipora DNA Samples from KBay Project

Goal Continue library prep for this project, resuspend final libraries in a larger volume
Results All libraries worked, the concentrations are high
Takeaways Looks like 35ul is a good volume to resuspend the final libraries in, the TS is not overloaded. Some of the peaks are broad but I don’t know how to troubleshoot that if it’s a problem

This library prep followed the closely the 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.

Dilution of samples to 10ng/ul in 10ul 20210513

Extraction-ID DNA reading Avg (ng/uL) Dilution for 100ng 10mM tris HCl to 10ul
42 80.2 1.25 8.75
6 24.5 4.08 5.92
46 88.6 1.13 8.87
17 32.9 3.04 6.96
40 40 2.50 7.50
34 37.15 2.69 7.31
35 32.1 3.12 6.88
16 36.5 2.74 7.26
50 69.3 1.44 8.56
37 33.9 2.95 7.05
23 35.7 2.80 7.20
33 89.4 1.12 8.88

Bisulfite Conversion

  • Followed exact steps as in protocol
  • Put samples in fridge overnight

Post-BS Conversion cleanup 20210510

  • 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 * 12.3 = 24.6ul
    • 1ul PrepAmp Primer * 12.3 = 12.3ul
  • PrepAmp Master Mix calculations (PAMM):
    • 1ul PrepAmp Buffer * 12.3 = 12.3ul
    • 3.75ul PrepAmp PreMix * 12.3 = 46.12ul
    • 0.3ul PrepAmp Polymerase * 12.3 = 3.69ul
  • Dilution calculation of PrepAmp Polymerase to add 0.5ul:
    • 0.3 PrepAmp Polymerase * 12.3 = 3.69ul
    • 0.2ul DNA elution buffer * 12.3 = 2.46ul

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 * 12.3 = 153.75ul
    • 1ul Library Amp Primers * 12.3 = 12.3ul

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 I used the KAPA HiFi HotStart Ready Mix instead of the Library Amp Mix and I used only 13ul of it, and that the indexes had been combined so I only used 1ul of the combined indexes
  • Table for components in tubes for amplifications:
Extraction-ID DNA Volume KAPA Mix Volume Primer Pair Volume Primer
42 12ul 13ul 13 1ul
6 12ul 13ul 14 1ul
46 12ul 13ul 15 1ul
17 12ul 13ul 16 1ul
40 12ul 13ul 17 1ul
34 12ul 13ul 18 1ul
35 12ul 13ul 19 1ul
16 12ul 13ul 20 1ul
50 12ul 13ul 21 1ul
37 12ul 13ul 22 1ul
23 12ul 13ul 23 1ul
33 12ul 13ul 24 1ul

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)
S1 179 RFU - -
S2 18882 RFU - -
42 25.4 25 25.2
6 31.6 31.6 31.6
46 33.6 33.2 33.4
17 35.6 35.4 35.5
40 21.8 21.6 21.7
34 31 30.4 30.7
35 29.2 29.2 29.2
16 35.4 35.8 35.6
50 30.4 30.4 30.4
37 31.4 31.4 31.4
23 21.6 21.4 21.5
33 31.8 31.8 31.8

D5000 TapeStation

Samples and Index Sequences

ColonyID Collection-Date Bleach Extraction-ID Index Pair (i5, i7) i7 bases i5 bases
M-222 2019-07-20 Non-Bleach 42 13 CCAAGTCT AAGGATGA
M-202 2019-12-04 Non-Bleach 6 14 TTGGACTC GGAAGCAG
M-202 2019-07-20 Non-Bleach 46 15 GGCTTAAG TCGTGACC
M-212 2019-12-04 Non-Bleach 17 16 AATCCGGA CTACAGTT
M-209 2019-07-20 Bleach 40 17 TAATACAG ATATTCAC
M-210 2019-12-04 Non-Bleach 34 18 CGGCGTGA GCGCCTGT
M-3 2019-12-04 Bleach 35 19 ATGTAAGT ACTCTATG
M-217 2019-12-04 Bleach 16 20 GCACGGAC GTCTCGCA
M-204 2019-07-20 Non-Bleach 50 21 GGTACCTT AAGACGTC
M-219 2019-07-20 Bleach 37 22 AACGTTCC GGAGTACT
M-219 2019-12-04 Bleach 23 23 GCAGAATT ACCGGCCA
M-218 2019-12-04 Non-Bleach 33 24 ATGAGGCC GTTAATTG
Written on May 14, 2021