Category Archives: Olympia Oyster Genome Sequencing

Phenol-Chloroform DNA Cleanup – Olympia Oyster gDNA

The gDNA I extracted on 20151104 didn’t look great on the NanoDrop so I decided to perform a phenol-chloroform cleanup to see if I could improve the NanoDrop1000 absorbance spectrum and, in turn, the quality of the gDNA.

  • Added an equal volume (500μL) of phenol:chloroform:isoamyl alcohol (25:24:1) to the DNA
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase to clean tube and discarded interphase & organic phase
  • Added an equal volume (380μL) of chlforoform:isoamyl alcohol (24:1)
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase (320μL) to clean tube
  • Added 0.1vols (32μL) of 3M sodium acetate (pH = 5.2)
  • Added 2vols (640μL) of 100% EtOH
  • Mixed by inversion
  • Incubated @ -20C, 1hr (probably not necessary since gDNA clearly precipitated out as soon as I mixed the sample)
  • Pelleted DNA by centrifuging 15mins, 12,000g, RT
  • Discarded supe
  • Washed pellet with 1000μL cold (-20C) 70% EtOH
  • Centrifuged 5mins, 12,000g, RT
  • Discarded supe
  • Repeated was steps three more times
  • Resuspended pellet in 100μL of Buffer EB (Qiagen)

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the 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:

 

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 547.15 200 109,430
Quant-IT 74.26 200 14,851

 

The NanoDrop1000 overestimates the concentration of the sample by 7.4x! That’s really insane!

Regardless, this is a solid yield (using yield from Quant-IT) and, when combined with the other Ostrea lurida gDNA that I isolated today, should push the total amount of gDNA submitted to BGI over the required threshold.

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

The clean up seems to have worked well, as the absorbance spectrum is much improved and nearly mirrors that of the Oly gDNA isolated with the Mollusc Kit.

DNA Isolation – Olympia Oyster Outer Mantle gDNA

Since we still don’t have sufficient gDNA for the full scope of the Olympia oyster genome sequencing, I isolated more gDNA.

Isolated gDNA from 118mg outer mantle tissue collected by Steven & Brent on 20150812.

Tissue was thoroughly minced with a clean razor blade and then processed with the E.Z.N.A. Mollusc Kit (Omega BioTek) with the following changes:

  • Doubled solution volumes for steps before sample was loaded on columns
  • Sample was split equally in two tubes prior to addition of 100% EtOH
  • All mixing was done by shaking – no vortexing! Done this way to, hopefully, maintain gDNA integrity
  • Elution volume = 50μL
  • Elution was repeated using the initial elution to maximize recovery while maintaining low sample volume.
  • The two preps were pooled – final volume = 79μL

DNA was quantified using two methods: NanoDrop1000 & QuantIT dsDNA BR Kit

For the Quant-IT kit, the 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:

METHOD CONCENTRATION (ng/μL) VOLUME (μL) YIELD (ng)
NanoDrop1000 552.53 79 43,650
Quant-IT 219.07 79 17,307

 

The NanoDrop1000 overestimates the concentration of the sample by 2.5x!

Regardless, this is a solid yield and, when combined with the other Ostrea lurida gDNA that I cleaned up today, should push the total amount of gDNA submitted to BGI over the required threshold.

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

 

DNA Quality Assessment – Geoduck, Oly & Oly 2SN

I recently ran gDNA isolated for geoduck and Olympia oyster genome sequencing, as well as gDNA isolated from the Olympia oyster reciprocal transplant experiment out on a Bioanalyzer (Agilent) using the DNA 12000 chips. The results from the chip were a bit confusing and difficult to assess exactly what was going on with the DNA.

So, I ran 5μL of each of those samples on a 0.8% agarose 1x modified TAE gel w/EtBr to get a better look at how the samples actually looked.

Results:

 

Both the geoduck and the Olympia oyster samples for genome sequencing show intact, high molecular weight bands with some smearing (i.e. degradation). The oly sample looks a bit funky, most likely due to a gel anomaly. I’ll quantify these using a dye-based method for a more accurate quantification before sending off to BGI.

The Fidalgo 2SN samples all have intact, high molecular weight bands, but most of the samples show extensive smearing (i.e. degradation). However, sample 2SN 35 has no visible DNA at all.

Here’s a table highlighting the differences between the Fidalgo gDNA samples:

Sample Fresh/Frozen Isolator
10 Fresh Sam
11 Fresh Sam
12 Fresh Sam
20 Fresh Mrunmayee
21 Fresh Mrunmayee
22 Fresh Mrunmayee
32 Frozen Sam
33 Frozen Sam
34 Frozen Sam
35 Frozen Sam

 

The fresh ctenidia samples were isolated by me on 20151021 and by Mrunmayee on 20151023. The frozen ctenidia samples were isolated by me on 20151103.

It’s interesting to note that Mrunmayee’s isolations appear to exhibit the least amount of degradation. Besides her handling the samples, the primary difference is that her samples were incubated in the buffer/Pro K solution O/N @ 37C, while my fresh samples were incubated @ 60C for 3hrs and my frozen samples were incubated @ 60C for 1hr. Overall, though, the frozen samples look the worst.

Finally, it’s also interesting to see that the two samples isolated using DNazol (geoduck and Olympia oyster genome samples) migrate more slowly than the remaining Olympia oyster samples which were isolated with the E.Z.N.A. Mollusc Kit.

DNA Quantification & Quality Assessment – Geoduck & Oly gDNA

Quantified the following samples with the Roberts Lab NanoDrop1000 (ThermoFisher) and assessed DNA integrity on the Seeb Lab Bioanalyzer 2100 (Agilent) using the DNA 12000 chip assay:

Results:

Bioanalyzer Data File (XAD file): 2100 expert_DNA 12000_DE72902486_2015-11-04_15-06-32.xad

 

OK, there’s a LOT going on here. Will update this notebook with my thoughts sometime tomorrow…

 

DNA Isolation – Geoduck & Olympia Oyster

Amazingly, we need more gDNA for the two genome sequencing projects (geoduck and Olympia oyster). Used geoduck adductor muscle sample from Box 1 of the geoduck samples collected by Brent & Steven on 20150811. Used Olympia oyster ctenidia from Box 1 of adductor muscle sample collected by Brent & Steven on 20150812.

Tissues were split in approximately half, minced and transferred to tubes with 1mL of DNAzol + 50μg/mL of Proteinase K (Fermentas). Previously, I had just homogenized samples. I’m hoping that the overnight digestion with Proteinase K will help increase yields from these.

Tissue weights:

  • Geoduck adductor muscle tube 1: 292mg (gone)
  • Geoduck adductor muscle tube 2: 320mg (gone)
  • Olympia oyster ctenidia tube 1: 135mg (gone)
  • Olympia oyster ctenidia tube 2: 130mg (gone)

Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

 

  • Samples were incubated O/N @ RT on a rotator.
  • After Proteinase K digestion, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended Buffer EB (Qiagen)

Resuspension volume = 500μL total for each species

Samples were incubated O/N at RT to facilitate pellet resuspension.

NOTE: Geoduck “pellets” were not very DNA pellet-like. Very loose, white, and sort of disintegrate (but not dissolve in solution) when attempted to resuspend.

Sample Submission – Additional Olympia Oyster gDNA for Genome Sequencing @ BGI

Previous shipment of gDNA proved to be of insufficient quantity when assessed by BGI, so needed to isolate more.

Shipped the pooled gDNA we’ve been accumulating to BGI to contine the geoduck genome sequencing project.

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

Assigned BGI Lot: 1510071002

Agarose Gel – Geoduck & Olympia Oyster gDNA

Needed to assess the integrity of the newest gDNA isolated for the two genome sequencing projects: Geoduck gDNA from earlier today and Olympia oyster gDNA from 20151002.

Also needed to assess the integrity of the gDNA of ethanol-preserved Olympia oyster mantle tissue from Jake’s reciprocal transplant experiment, isolated on 20151002: samples NF1A & SN49A.

Ran samples on a 0.8% agarose, 1X modified TAE gel.

Loaded 1μL (~300ng) of the geoduck, Oly and NF1A samples.

Loaded 2μL (~100ng) of the SN49A sample.

Used 5μL of ladder.

Results:

 

Genome Sequencing Samples

The geoduck and the Oly samples look good. Intact, high molecular weight band. My only concern is the noticeable difference in band intensities between these two. Both samples should be ~300ng/μL, based on the NanoDrop1000 readings. However, it’s evident that the concentrations of these two samples differ greatly. Additionally, we can use the ladder to gauge the concentrations of the samples, since I loaded 0.5μg of the ladder, which is the quantity referenced on the ladder guide above.

It would appear that the geoduck sample concentration is closer to 60ng/μL (band intensity is similar to that of the 500, 1000, & 3000bp markers), as opposed to the 292ng/μL that the NanoDrop1000 indicated.

The Oly sample appears to have even less and appears less intense than the lowest concentration bands on the marker (16.0ng/μL). That’s not even remotely close to the 331ng/μL measured by the NanoDrop1000.

It’s difficult to say why this might be, as both samples were RNased and neither of them show extensive smearing (both of those factors would contribute to inflated spec readings).

Regardless, will ship them off to BGI to supplement the previous gDNA for this project.

Ethanol-Preserved Samples

Both samples show extensive smearing and no high molecular weight band, indicating they are both completely degraded. This is a very bad result for this project, as the tissue in this group is/was a bit of grasping at straws to obtain some intact DNA to use for the RAD-seq that we intend to pursue.

DNA Isolation – Geoduck & Olympia Oyster

Amazingly, we need more gDNA for the two genome sequencing projects (geoduck and Olympia oyster). Used geoduck “foot 1″ sample from Box 1 of the foot samples collected by Brent & Steven on 20150811. Used Olympia oyster adductor muscle from Box 1 of adductor muscle sample collected by Brent & Steven on 20150812.

Also need to evaluate DNA quality of initial broodstock samples from Jake’s Olympia oyster reciprocal transplant experiment. Used mantle samples stored in EtOH collected by Hannah (see her notebook entries on July 25 & Sept 5, 2013)

Tissue weights:

  • Geoduck foot: 108.5mg (gone)
  • Olympia oyster adductor: 258.7mg (gone)
  • Oly NF1A: 7.1mg (gone)
  • Oly SN49A: 20.8mg

 

Samples were isolated using DNAzol (Molecular Research Center) according to the manufacturer’s protocol, with the following adjustments:

 

  • Tissues homogenized in 750μL of DNAzol with disposable mortar/pestle tubes using 10 pestle strokes
  • After homogenization, topped off tubes to 960μL with DNAzol, added 40μL RNAse A (100mg/mL) and incubated @ RT for 15mins.
  • Performed optional centrifugation step (10,000g, 10mins @ RT)
  • Initial pellet wash was performed using a 70%/30% DNAzol/EtOH
  • Pellets were resuspended Buffer EB (Qiagen)
  • Insoluble material was pelleted (12,000g, 10mins @ RT) and supe transferred to new tubes

 

Genome sequencing resuspension volumes: 50μL

Oly reciprocoal resuspension volumes: 25μL

Spec’d on Roberts Lab NanoDrop1000.

Results:

 

Genome Sequencing Samples

The 260/280 ratios look fine. The 260/230 ratios look poor, as is usually the case after DNAzol isolations.

Yields:

Geoduck: 7.6μg

Oly: 16.5μg

The geoduck yield is insufficient to make up the quantity of gDNA still needed by BGI for sequencing. Will have to isolate more gDNA on Monday.

 

Reciprocal Transplant Samples

The 260/280 ratios look fine. The 260/230 ratios look poor, as is usually the case after DNAzol isolations.

Yields:

NF1A: 7,1μg

SN49A: 1.375μg

The yields are surprisingly good! Next up is to evaluate the gDNA quality on a gel to see if the samples from this experiment will be usable.

Sample Submission – Olympia Oyster gDNA for Genome Sequencing @ BGI

Shipped the pooled gDNA we’ve been accumulating to BGI to initiate the Olympia oyster genome sequencing project.

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

Assigned BGI Lot: 1509191001

Agarose Gel – Geoduck & Olympia oyster gDNA Integrity Check

Ran a 0.8% agarose, 1x modified TAE gel (w/EtBr) with geoduck and Olympia oyster gDNA that was precipitated earlier today. Used 5μL of each sample (~500ng).

Results:

Geoduck gDNA on left. Oly gDNA on right.

 

 

 

 

 

 

 

 

 

 

 

 

 

Overall, the DNA still looks very good. Slight smearing (indicating slight degradation), but the high molecular weight band is very prominent. Will fill out the necessary BGI forms and ship samples out on Monday.