Received the Olympia oyster, MBD-enriched BS-seq sequencing files (50bp, single read) from ZymoResearch (submitted 20151208). Here’s the sample list:

• E1_hc1_2B
• E1_hc1_4B
• E1_hc2_15B
• E1_hc2_17
• E1_hc3_1
• E1_hc3_5
• E1_hc3_7
• E1_hc3_10
• E1_hc3_11
• E1_ss2_9B
• E1_ss2_14B
• E1_ss2_18B
• E1_ss3_3B
• E1_ss3_14B
• E1_ss3_15B
• E1_ss3_16B
• E1_ss3_20
• E1_ss5_18

The 18 samples listed above had previously been MBD-enriched and then sent to ZymoResearch for bisulfite conversion, multiplex library construction, and subsequent sequencing. The library (multiplex of all samples) was sequenced in a single lane, three times. Thus, we would expect 54 FASTQ files. However, ZymoResearch was dissatisfied with the QC of the initial sequencing run (completed on 20160129), so they re-ran the samples (completed on 20160202). This created two sets of data, resulting in a total of 108 FASTQ files.

ZymoResearch data portal does not allow bulk download of files. However, I ended up using Chrono Download Manager extension for Google Chrome to allow for automated downloading of each file (per ZymoResearch recommendation).

After download, the files were moved to their permanent storage location on Owl: http://owl.fish.washington.edu/nightingales/O_lurida/20160203_mbdseq

The readme.md file was updated to include project/file information.

The file manipulations were performed in a Jupyter notebook (see below).

Total reads generated for this project: 1,481,836,875

Jupyter Notebook file: 20160203_Olurida_Zymo_Data_Handling.ipynb

Notebook Viewer: 20160203_Olurida_Zymo_Data_Handling.ipynb

We opted to go with ZymoResearch for this project because they could do the bisulfite treatment and finish the sequencing by the end of January.

Submitted the following 18 Ostrea lurida MBD-enriched gDNA samples to ZymoResearch for bisulfite treatment and subsequent Illumina sequencing (50bp, single read):

The samples will be bisulfite treated, Illumina libraries constructed, multiplexed, and this multiplexed library will be sequenced three times.

Olympia oyster gDNA that had previously been sonicated and fragmented was enriched for the methylated fragments using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen).

Prepared the following components:

• 20mL 1x Bind/Wash Buffer (4mL 5x Bind/Wash Buffer + 16mL H2O)
• 640μL of beads (35μL of beads x 18 samples )
• 200μL MBD-Biotin Protein (63μL MBD-Biotin Protein + 137μL 1x Bind/Wash Buffer)

Followed the manufacturer’s protocol for input DNA quantities 1μg – 10μg.

Used single fraction, high salt elution.

Neglected to account for the control reaction during initial set up and did not have sufficient quantities of beads to run a control reaction.

The table below provides the individual sample volumes and the volumes of the buffer, beads, H2O for the MBD capture reactions.

Samples listed with “NA” were not processed because they did not fragment during sonication.

Non-captured & wash fractions were pooled into single samples and stored @ -20C.

MBD fraction was EtOH precipitated according to the manufacturer’s protocol and incubate O/N @ -80C.

In preparation for MBD enrichment, fragmented Olympia oyster gDNA with a target size of ~350bp.

Genomic DNA samples were isolated and provided to us by Katherine Silliman at UIC. Selected samples will compare Hood Canal (HC) and Oyster Bay (SS, South Sound) populations.

Used the Seeb Lab’s Bioruptor 300 (Diagenode) sonicator.

After sonication, samples were run on a the Seeb Lab’s 2100 Bioanalyzer (Agilent) on DNA 12000 chips.

Results:

HOOD CANAL SAMPLES

OYSTER BAY SAMPLES

More detailed analysis (including average fragment size for each samples) will be coming soon…