Tag Archives: bisulfite conversion

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.

BS-seq Library Prep – C.gigas Larvae OA 1000ppm

Bisulfite Conversion

Pooled 200ng each of the sheared 1B1 (4μL) & 1B2 (used the entire sample, 20μL) 5.13.11 1000ppm C.gigas larvae DNA samples for a total of 400ng. Total volume = 24μL.

Quantified the pooled DNA using the NanoDrop1000 (ThermoFisher) prior to initiating bisulfite conversion.

Clearly, the NanoDrop measurements differ from the expected concentration. NanoDrop suggests the total amount of input DNA is ~1400ng (58ng/μL x 24μL = 1392ng). This is most likely due to RNA carryover, as DNA quantification using a fluorescence-based, double-stranded DNA assay performed previously shows a drastically lower concentration.

Proceeded with bisulfite conversion using the Methylamp DNA Modification Kit (Epigentek) in 1.5mL tube, according to the manufacturer’s protocol:

  • Added 1μL to sample, incubated 10mins @ 37C in water bath
  • Made fresh R1/R2/R3 solution (1.1mL R3 buffer added to vial of R2, vortexed 2mins, 40μL R1 added to mixture – Remainder stored @ -20C in “-20C Kit Components Box”)
  • Added 125μL of R1/R2/R3 solution to sample, incubated 90mins @ 65C in heating block with water
  • Addd 300μL R4 to sample, mixed, transferred to column, spun 12,000RPM 30s
  • Added 200μL R5 to column, spun 12,000RPM 30s
  • Added 50μL R1/ethanol solution to column, incubated 8mins @ RT, spun 12,000RPM 30s
  • Washed column with 200μL of 90% EtOH, spun 12,000RPM 30s; repeated one time.
  • Eluted DNA with 12μL R6, spun 12,000RPM 30s

Quantified post-bisulfite-treated sample on NanoDrop1000:

Definitely a low yield (~108ng) relative to the input (~400ng). Will proceed with Illumina library prep.

 

Library Prep

Illumina library prep was performed with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • PCR cycles: 15

No other changes were made to the manufacturer’s protocol.

Epigentek Barcode Indices assigned, per their recommendations for using two libraries for multiplexing (this will be combined with the 400ppm library):

Barcode #12 – CTTGTA

The library was stored @ -20C and will be checked via Bioanalyzer on Monday.

Bisulfite NGS Library Prep – Bisulfite Conversion & Illumina Library Construction of C.gigas larvae DNA

Bisulfite Conversion

The previous attempt at constructing a library for the 1000ppm larvae samples failed. I had previously sheared, quantified, and concentrated the DNA from this sample. As I had done previously, I combined 50ng from each of the two 1000ppm samples for a total of 100ng, and brought the sample volume up to 24μL with NanoPure H2O.

Bisulfite conversion was performed with the Methylamp DNA Modification Kit (Epigentek) according to the manufacturer’s protocol.

Sample was eluted with 10μL of Buffer R6 for immediate use.

 

Library Prep

Bisulfite Illumina library was made with EpiNext Post-Bisulfite DNA Library Preparation Kit (Illumina) (Epigentek).  Changes to the manufacturer’s protocol:

  • Samples were transferred to 1.5mL snap cap tubes for all magnetic bead steps in order to fit in our tube magnets.
  • Skipped Step 7.1 (per manufacturer’s recommendation for samples starting with <200ng)
  • Ran 13 cycles during the library amplification step (per manufacturer’s recommendation for samples starting with 100ng)

Sample was transferred to 1.5mL snap cap tube and stored @ -20C.  Will quantify library on Monday when Jake is also finished with his 12 libraries.

 

DNA Bisulfite Conversion – C.gigas larvae OA Sheared DNA

After discussing with Steven, decided to use only samples from 20110513, due to high algae amounts present in the 20110519 samples.

Pooled the DNA of the 400ppm samples (6B2 & 6B5) and pooled the DNA of the 1000ppm samples(1B1 & 1B2) , for a total of two samples. 50ng of each sample was used to make the pools, for a toal of 100ng of DNA in each pool. Calculations are below:
https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

Each pooled sample was brought up to a final volume of 24μL for use in bisulfite conversion kit.

Performed bisulfite conversion of sheared DNA from 20150113 using the Methylamp DNA Modification Kit (Epigentek) according the manufacturer’s protocol.

Samples were eluted with 10μL of Buffer R6 and stored @ -20C.

 

Bisulfite Conversion – LSU C.virginica Oil Spill MBD DNA and Emma’s C.gigas Larvae OA DNA

Performed bisulfite conversion on MBD DNA samples from LSU C.virginica oil spill samples (see 201411202 and 20141126) and Emma’s C.gigas larvae OA DNA samples (see 20141121) with the Methylamp DNA Modification Kit (Epigentek).

Added 4uL of H2O to each of Emma’s DNA samples to bring them up to 24uL.

Samples were processed according to the manufacturer’s protocol.

Samples were eluted with 10uL of Solution R6 and stored @ -20C.