Pooled 10ng of each of the libraries prepared yesterday with TruSeq DNA Methylation Library Kit (Illumina) for sequencing at Genewiz.
| SAMPLE | VOLUME FOR 10ng (μL) |
| 1NF11 | 4.13 |
| 1NF15 | 5.32 |
| 1NF16 | 3.65 |
| 1NF17 | 3.94 |
| 2NF4 | 3.68 |
| 2NF5 | 4.10 |
| 2NF6 | 4.20 |
| 2NF7 | 5.32 |
| M2 | 4.59 |
| M3 | 3.91 |
| NF2_6 | 4.00 |
| NF_18 | 3.76 |
Samples were sent to Genewiz on dry ice via standard overnight FedEx.
Re-quantified the libraries that were completed yesterday using the Qubit3.0 dsDNA HS (high sensitivity) assay because the library concentrations were too low for the normal broad range kit.
Results:
Qubit Quants and Library Normalization Calcs: 20151222_qubit_illumina_methylation_libraries
| SAMPLE | CONCENTRATION (ng/μL) |
| 1NF11 | 2.42 |
| 1NF15 | 1.88 |
| 1NF16 | 2.74 |
| 1NF17 | 2.54 |
| 2NF5 | 2.72 |
| 2NF6 | 2.44 |
| 2NF7 | 2.38 |
| 2NF8 | 1.88 |
| M2 | 2.18 |
| M3 | 2.56 |
| NF2_6 | 2.5 |
| NF_18 | 2.66 |
Things look pretty good. The TruSeq DNA Methylation Library Kit (Illumina) suggests that the libraries produced should end up with concentrations >3ng/μL, but we have plenty of DNA here to make a pool for running on the HiSeq2500.
Took the bisulfite-treated DNA from 20151218 and made Illumina libraries using the TruSeq DNA Methylation Library Kit (Illumina).
Quantified the completed libraries using the Qubit 3.0 dsDNA BR Kit (ThermoFisher).
Evaluated the DNA with the Bioanalyzer 2100 (Agilent) using the DNA 12000 assay. Illumina recommended using the High Sensitivity assay, but we don’t have access to that so I figured I’d just give the DNA 12000 assay a go.
| SampleName | IndexNumber | BarCode |
| 1NF11 | 1 | ATCACG |
| 1NF15 | 2 | CGATGT |
| 1NF16 | 3 | TTAGGC |
| 1NF17 | 4 | TGACCA |
| 2NF5 | 5 | ACAGTG |
| 2NF6 | 6 | GCCAAT |
| 2NF7 | 7 | CAGATC |
| 2NF8 | 8 | ACTTGA |
| M2 | 9 | GATCAG |
| M3 | 10 | TAGCTT |
| NF2_6 | 11 | GGCTAC |
| NF_18 | 12 | CTTGTA |
Results:
Library Quantification (Google Sheet): 20151221_quantification_illumina_methylation_libraries
| Test Name | Concentration (ng/μL) |
| 1NF11 | Out of range |
| 1NF15 | 2.14 |
| 1NF16 | 2.74 |
| 1NF17 | 2.64 |
| 2NF5 | 2.92 |
| 2NF6 | Out of range |
| 2NF7 | 2.42 |
| 2NF8 | 2.56 |
| M2 | Out of range |
| M3 | 2.1 |
| NF2_6 | 2.38 |
| NF2_18 | Out of range |
I used the Qubit’s BR (broad range) kit because I wasn’t sure what concentrations to expect. I need to use the high sensitivity kit to get a better evaluation of all the samples’ concentrations.
Bioanalyzer Data File (Bioanalyzer 2100): 2100_20expert_DNA_2012000_DE72902486_2015-12-21_16-58-43.xad
Ha! Well, looks like you definitely need to use the DNA High Sensitivty assay for the Bioanalyzer to pick up anything. Although, I guess you can see a slight hump in most of the samples at the appropriate sizes (~300bp); you just have to squint. 
Following the guidelines of the TruSeq DNA Methylation Library Prep Guide (Illumina), I ran 1μL of each sample on an RNA Pico 6000 chip on the Seeb Lab’s Bioanalyzer 2100 (Agilent) to confirm that bisulfite conversion from earlier today worked.
Results:
Data File 1(Bioanlyzer 2100): 2100 expert_Eukaryote Total RNA Pico_DE72902486_2015-12-18_21-05-04.xad
Data File 1(Bioanlyzer 2100): 2100 expert_Eukaryote Total RNA Pico_DE72902486_2015-12-18_21-42-55.xad
Firstly, the ladder failed to produce any peaks. Not sure why this happened. Possibly not denatured? Seems unlikely, but next time I run the Pico assay, I’ll denature the ladder aliquot I use prior to running.
Overall, the samples look as they should (see image from TruSeq DNA Methylation Kit manual below), albeit some are a bit lumpy.
After confirming that the DNA available for this project looked good, I performed bisulfite treatment on the following gDNA samples:
Sample names breakdown like this:
1NF#
1 = Fidalgo Bay outplants
NF = Fidalgo Bay broodstock origination
# = Sample number
2NF#
Same as above, but:
2 = Oyster Bay outplants
NF2_# (Oysters grown in Oyster Bay; DNA provided by Katherine Silliman)
NF2 = Fidalgo Bay broodstock origination, family #2
# = Sample number
M2/M3 = C.gigas from Katie Lotterhos
Followed the guidelines of the TruSeq DNA Methylation Library Prep Guide (Illumina).
Used the EZ DNA Methylation-Gold Kit (ZymoResearch) according to the manufacturer’s protocol with the following changes/notes:
DNA quantity calculations are here (Google Sheet): 20151218_oly_bisulfite_calcs
Samples were stored @ -20C. Will check samples via Bioanalyzer before proceeding to library construction.