Steven asked me to evaluate our methylation sequencing data sets for Olympia oyster.

According to our Olympia oyster genome wiki, we have the following two sets of BS-seq data:

• Whole genome BS-seq (2015)

• MBD BS-seq (2015)

All computing was conducted on our Apple Xserve: emu.

All steps were documented in this Jupyter Notebook (GitHub): 20180503_emu_oly_methylation_mapping.ipynb

NOTE: The Jupyter Notebook linked above is very large in size. As such it will not render on GitHub. It will need to be downloaded to a computer that can run Jupyter Notebooks and viewed that way.

Here’s a brief overview of what was done.

Samples were trimmed with TrimGalore and then evaluated with FastQC. MultiQC was used to generate a nice visual summary report of all samples.

The Olympia oyster genome assembly, pbjelly_sjw_01, was used as the reference genome and was prepared for use in Bismark:

/home/shared/Bismark-0.19.1/bismark_genome_preparation \
--path_to_bowtie /home/shared/bowtie2-2.3.4.1-linux-x86_64/ \
--verbose /home/sam/data/oly_methylseq/oly_genome/ \
2> 20180507_bismark_genome_prep.err

Bismark was run on trimmed samples with the following command:

/home/shared/Bismark-0.19.1/bismark \
--path_to_bowtie /home/shared/bowtie2-2.3.4.1-linux-x86_64/ \
--genome /home/sam/data/oly_methylseq/oly_genome/ \
-u 1000000 \
-p 16 \
--non_directional \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/1_ATCACG_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/2_CGATGT_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/3_TTAGGC_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/4_TGACCA_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/5_ACAGTG_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/6_GCCAAT_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/7_CAGATC_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/8_ACTTGA_L001_R1_001_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_10_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_11_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_12_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_13_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_14_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_15_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_16_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_17_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_18_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_1_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_2_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_3_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_4_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_5_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_6_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_7_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_8_s456_trimmed.fq.gz \
/home/sam/analyses/20180503_oly_methylseq_trimgalore/zr1394_9_s456_trimmed.fq.gz \
2> 20180507_bismark_02.err

Results:

TrimGalore output folder:

• 20180503_oly_methylseq_trimgalore

FastQC output folder:

• 20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/

MultiQC output folder:

• 20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/multiqc_data/

MultiQC Report (HTML):

• 20180503_oly_methylseq_trimgalore/20180503_trim_fastqc/multiqc_data/multiqc_report.html

Bismark genome folder: 20180503_oly_genome_pbjelly_sjw_01_bismark/

Bismark output folder:

• 20180507_oly_methylseq_bismark

Whole genome BS-seq (2015)

Prep overview

• Library prep: Roberts Lab
• Sequencing: Genewiz

MBD BS-seq (2015)

Prep overview

• MBD: Roberts Lab
• Library prep: ZymoResearch
• Sequencing: ZymoResearch

Steven requested I concatenate the MBDseq files we received for this project:

• concatenate the s4, s5, s6 file sets for each individual

• concatenate the full file sets for each individual

Ran the concatenations in the Jupyter (iPython) notebook below. All files were saved to Owl/nightingales/O_lurida/2016

Jupyter Notebook: 20160411_Concatenate_Oly_MBDseq.ipynb

NBviewer: 20160411_Concatenate_Oly_MBDseq

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.

Quantified the MBD enriched samples prepped over the last two days: MBD enrichment, EtOH precipiation.

Samples were quantified using the QuantIT dsDNA BR Kit (Invitrogen) according to the manufacturer’s protocol.

Standards were run in triplicate, samples were run in duplicate.

96-well black (opaque) plate was used.

Fluorescence was measured on the Seeb Lab’s Victor 1420 plate reader (Perkin Elmer).

Results:

Google Sheet: 20151123_MBD_libraries_quantification

Standard curve looked good – R² = 0.999

MBD recovery ranged from ~250 – 600ng.

MBD percent recoveries ranged from ~2 – 20%. Input DNA quantities were taken from Katherine’s numbers (Google Sheet): Silliman-DNA-Samples

Will contact services about getting bisulfite Illumina sequencing performed.

Precipitated the MBD enriched DNA from yesterday according to the MethylMiner Methylated DNA Enrichment Kit (Invitrogen) protocol.

However, since the protocol has two elution steps that are each saved separately from each other for each sample, I did the following to combine the two elution fractions into a single sample:

• Pelleted one elution fraction from each sample
• Discarded supernatant from pelleted sample
• Transferred second elution fraction to the pellet from the first elution fraction
• Pelleted second elution fraction

The rest of the ethanol precipitation procedure was followed per the manufacturer’s protocol.

Final pellets were resuspended in 25μL of Buffer EB (Qiagen) and stored @ 4C.

MBD enriched DNA will be quantified tomorrow.

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…