Tag Archives: NF2_6

Illumina Methylation Library Quantification – BS-seq Oly/C.gigas Libraries

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.

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Illumina Methylation Library Construction – Oly/C.gigas Bisulfite-treated DNA

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. ;)

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Bioanalyzer – Bisulfite-treated Oly/C.gigas DNA

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.

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Bisulfite Treatment – Oly Reciprocal Transplant DNA & C.gigas Lotterhos DNA for BS-seq

After confirming that the DNA available for this project looked good, I performed bisulfite treatment on the following gDNA samples:

  • 1NF11
  • 1NF15
  • 1NF16
  • 1NF17
  • 2NF5
  • 2NF6
  • 2NF7
  • 2NF8
  • NF2_6
  • NF2_18
  • M2
  • M3

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:

  • Used 100ng DNA (per Illumina recs; Zymo recommends at least 200ng for “optimal results”).
  • Thermal cycling was performed in 0.5mL thin-wall tubes in a PTC-200 (MJ Research) using a heated lid
  • Centrifugations were performed at 13,000g
  • Desulphonation incubation for 20mins.

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.

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