Ran the 400ppm library and the 1000ppm library preps on a DNA1000 Assay Chip (Agilent) on the Agilent 2100 Bioanalyzer.

Results:

Data File (XAD): 2100_expert_DNA_1000_DE72902486_2015-03-02_09-18-02.xad

Electropherogram overlay of both samples:

Red = 400ppm

Blue = 1000ppm

Measurement data and parameters are here: 20150302_Bioanalyzer_Cgigas_400_1000ppm_BS-Seq

Both libraries look good; no adaptor contamination (peak would be present at ~125bp), good library sizes.

Pooled equal quantities of each library, based off the concentration values above, to prepare the sample for sequencing.

The pooled libraries will be submitted tomorrow to the Genomics Core Facility at the Univ. of Oregon for high-throughput sequencing (100bp, SE) on the HiSeq2500 (Illumina). Sample order #2212.

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.