Sent Crassostrea virginica samples to Qiagen, as part of the collaboration we have with them for testing their new bisulfite conversion kit on various reduced representation DNA.

Samples were sent on dry ice via FedEx International Priority: 771231112481

Here are the samples I sent:

gDNA C.virginica – Genomic DNA, 20uL, 58.4ng/uL
MBD 1 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 18.3ng/uL
MBD 2 virginica – Fragmented (~400bp average size), MBD-enriched, 25uL, 19.6ng/uL
MspI 1 virginica – gDNA digested with MspI, 25uL, 53.4ng/uL
MspI 2 virginica – gDNA digested with MspI, 25uL, 31.0ng/uL

Genomic DNA was isolated from mantle tissue using the E.Z.N.A. Mollusc DNA Kit (Omega). DNA was eluted with the Elution Buffer supplied with the kit.

MBD enrichment was performed using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen). DNA was resuspended in Buffer EB (Qiagen).

MspI digestions were performed using MspI (NEB) and were subjected to a phenol:chloroform cleanup, post-digestion. DNA was resuspended in Buffer EB (Qiagen).

No tests have been performed on the samples to evaluate the presence/absence of non-oyster DNA.

Reference genome is available here: https://www.ncbi.nlm.nih.gov/assembly/GCF_002022765.2/

Notebook entries:

gDNA Isolation:

http://onsnetwork.org/kubu4/2017/12/11/dna-isolation-quantification-crassotrea-virginica-mantle-gdna/

MBD:

DNA Shearing and Bioanalyzer assessments: http://onsnetwork.org/kubu4/2017/12/11/dna-sonication-bioanalzyer-c-virginica-gdna-for-medip/
Day 1: http://onsnetwork.org/kubu4/2018/01/08/mbd-enrichment-crassostrea-virginica-sheared-dna-day-1/
Day 2: http://onsnetwork.org/kubu4/2018/01/09/mbd-enrichment-crassostrea-virginica-sheared-dna-day-2/
Day 3: http://onsnetwork.org/kubu4/2018/01/10/mbd-enrichment-crassostrea-virginica-sheared-dna-day-3/
Quants: http://onsnetwork.org/kubu4/2018/01/10/dna-quantification-c-virginica-mbd-enriched-dna/

MspI Digestion:

Digestion: http://onsnetwork.org/kubu4/2018/01/11/restriction-digestion-mspi-on-crassotrea-virginica-gdna/
Phenol:Chloroform cleanup: http://onsnetwork.org/kubu4/2018/01/11/phenolchloroform-extractions-and-etoh-precipitations-mspi-digestions-of-c-virginica-dna-from-earlier-today/
Quants: http://onsnetwork.org/kubu4/2018/01/11/dna-quantification-mspi-digested-crassostrea-virginica-gdna/

I isolated DNA from the Crassotrea virginica gonad samples sent by Katie Lotterhos using the E.Z.N.A. Mollusc Kit with the following modifications:

• Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
• No optional steps were used
• Eluted each in 100μL of Elution Buffer and pooled into a single sample

NOTE: Sample 034 did not process properly (no phase separation after 24:1 chlorform:IAA addition – along with suggested additions of ML1 Buffer) and was discarded.

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 2μL of DNA sample.

Samples were stored in the same box the tissue was delivered in and stored in the same location in our -80C: rack 8, row 5, column 4.

Results:

Qubit (Google Sheet): 20171114_qubit_Cvirginica_gDNA

Ample DNA in all samples for MBDseq. (Refer to “Original Sample Conc.” column in spreadsheet.)

Will let Steven & Katie know.

Sent 10μg of the geoduck gDNA I isolated earlier today to Illumina on dry ice via FedEx Standard Overnight service.

We were previously approached by Cindy Lawley (Illumina Market Development) for possible participation in an Illumina product development project, in which they wanted to have some geoduck tissue and DNA on-hand in case Illumina green-lighted the use of geoduck for testing out the new sequencing platform on non-model organisms. Well, guess what, Illumina has give the green light for sequencing our geoduck! However, they need at least 4μg of gDNA, so I’m isolating more.

Isolated DNA from ctenidia tissue from the same Panopea generosa individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150811.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from five separate ~60mg pieces of ctenidia tissue according to the manufacturer’s protocol, with the following changes:

• Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
• Incubated homogenate at 60C for 1hr
• No optional steps were used
• Performed three rounds of 24:1 chloroform:IAA treatment
• Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 162ng/μL (Quant data is here [Google Sheet]: 20170105_gDNA_geoduck_qubit_quant

Yield is great (total = ~32μg).

Evaluated gDNA quality (i.e. integrity) by running 162ng (1μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

Results:

DNA looks good: bright high molecular weight band, minimal smearing, and minimal RNA carryover (seen as more intense “smear” at ~500bp).

Will send off 10μg (they only requested 4μg) so that they have extra to work with in case they come across any issues.

Submitted 10μg (30.7μL) of the O.lurida gDNA I isolated on 20161214 to the UW PacBio facility – Order #450.

Sequencing will be 10 SMRT cells. Turnaround time is ~7-8 weeks for UW customers (UW customers get queue priority).

In an attempt to improve upon the partial genome assembly we received from BGI, we will be sending DNA to the UW PacBio core facility for additional sequencing.

Isolated DNA from mantle tissue from the same Ostrea lurida individual used for the BGI sequencing efforts. Tissue was collected by Brent & Steven on 20150812.

Used the E.Z.N.A. Mollusc Kit (Omega) to isolate DNA from two separate 50mg pieces of mantle tissue according to the manufacturer’s protocol, with the following changes:

• Samples were homogenized with plastic, disposable pestle in 350μL of ML1 Buffer
• Incubated homogenate at 60C for 1.5hrs
• No optional steps were used
• Performed three rounds of 24:1 chloroform:IAA treatment
• Eluted each in 50μL of Elution Buffer and pooled into a single sample

Quantified the DNA using the Qubit dsDNA BR Kit (Invitrogen). Used 1μL of DNA sample.

Concentration = 326ng/μL (Quant data is here [Google Sheet]: 20161214_gDNA_Olurida_qubit_quant

Yield is good and we have more than enough (~5μg is required for sequencing) to proceed with sequencing.

Evaluated gDNA quality (i.e. integrity) by running ~500ng (1.5μL) of sample on 0.8% agarose, low-TAE gel stained with ethidium bromide.

Used 5μL of O’GeneRuler DNA Ladder Mix (ThermoFisher).

Results:

Overall, the gel looks OK. A fair amount of smearing, but a strong, high molecular weight band is present. The intensity of the smearing is likely due to the fact that the gel is overloaded for this particular well size. If I had used a broader comb and/or loaded less DNA, the band would be more defined and the smearing would be less prominent.

Will submit sample to the UW PacBio facility tomorrow!

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.

Ran 1μL of each sample from yesterday’s DNA isolation on a 0.8% agarose, low-TAE gel, stained with ethidium bromide.

Results:

Since I didn’t load equal quantities of DNA, the intensities across the various samples is highly variable.

Those samples with high degree of smearing are also those with the highest concentrations. Thus, one would expect to be able to visualize a greater range of DNA sizes in a gel (because more DNA is present). Notice the samples with nice, high molecular weight bands and little smearing (1NF16, 1NF17). These are less than half the concentrations of all the samples that exhibit extensive smearing (2NF3, 2NF8, 1NF12). So, I think all samples will be fine for proceeding with bisulfite conversion and subsequent library construction.

However, I should re-run this gel using equalized DNA quantities for all samples…

Received 6 samples from Katie today. The box was labeled and stored @ -20C.

Here description of the samples, via email:

Yep, BGI still needs more gDNA for the Olympia oyster genome sequencing project. Samples have been quantified via dye-based fluorescence, as opposed to the NanoDrop, so our quantities should be more accurate and in-line with what BGI will also find.

Submitted three separate isolations, just in case the quality of one was unacceptable, I didn’t want to pool the samples and have that one bad apple ruin the entire batch.

In total, submitted ~31μg.

Samples were shipped on dry ice with the appropriate paperwork required by BGI (sample declaration letter).

Assigned BGI Lot: 1512021005