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

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

Sample Submission – Additional Geoduck gDNA for Genome Sequencing @ BGI

Yep, BGI still needs more gDNA for the geoduck 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 ~33μg.

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

Assigned BGI Lot: 1512021004

 

Data Storage – Synology DX513

Running a bit low on storage on Owl (Synology DS1812+) and we will be receiving a ton of data in the next few months, so we purchased a Synology DX513. It’s an expansion unit designed specifically for seamlessly expanding our existing storage volume on Owl.

Installed 5 x 8TB Seagate HDDs and connected to Owl with the supplied eSATA cable.

Now, we just need to wait (possibly days) for the full expansion to be completed.

Sample Submission – Oly Oyster Bay Tissues for GBS

Sent Olympia oyster tissue samples to BGI for genotype-by-sequencing (GBS). Tissues were shipped FedEx standard overnight on dry ice.

Tissues were collected by Brent, Steven & Jake on 20151124 and were frozen @ -80C.

36 samples from each of the three populations were collected. Only 32 samples from each population (total 96 samples) will be sequenced, but wanted to send extras from each population in case any were of poor quality.

Sample submission sheet is here (Google Sheet): 20151130_BGI_GBS_tissue_submission

DNA Quality Assessment – Geoduck & Olympia Oyster gDNA

Have three separate sets of geoduck & olympia oyster gDNA that need to be run on gels before sending to BGI for genome sequencing:

GEODUCK

 

OLYMPIA OYSTER

 

Ran 100ng of each sample on a 0.8% agarose 1x modified TAE gel w/EtBr.

Results:

 

All the samples from both sets appear to be overloaded. Overloading is generally seen as the streaking seen immediately above each band.

GEODUCK

Overall, the samples look pretty good. Sadly, the worst of the three (due to the most smearing – i.e. degradation) appears to be the DNA extracted using the E.Z.N.A. Mollusc Kit (Omega BioTek).

Also of note are the two bands present in the DNAzol sample. These bands are likely ribosomal RNA because I neglected to perform a RNase treatment during the extraction. Doh!

 

OLYMPIA OYSTER

None of them are particularly great. Just like the geoduck set, the worst of the three came from the E.Z.N.A Mollusc Kit (Omega BioTek).

Also, just like the geoduck set, there are two bands present in the DNAzol sample. These bands are likely ribosomal RNA because I neglected to perform a RNase treatment during the extraction. Doh!

The phenol-chloroform clean up sample is either jacked up or severely overloaded, based on the crazy streaking that’s present. However, this sample looked similar after the initial extraction on 20151113.

 

I will send these samples separately (i.e. will not pool them into single samples) to BGI to run QC and, hopefully, add them to the DNA they already have to complete the genome sequencing for these two projects.

DNA Isolation – Olympia Oyster Outer Mantle gDNA

Isolated additional gDNA for the genome sequencing. To try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.

Used 123mg of Ostrea lurida outer mantle collected by Brent & Steven on 20150812.

  • Homogenized in 500μL of DNAzol.
  • Added additional 500μL of DNAzol.
  • Centrifuged 12,000g, 10mins, @ RT.
  • Split supernatant equally into two tubes.
  • Added 500μL of chloroform and mixed moderately fast by hand.
  • Centrifuged 12,000g, 10mins, RT.
  • Combined aqueous phases from both tubes in a clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Spooled precipitated gDNA and transferred to clean tube.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 200μL of Buffer EB (Qiagen).
  • Centrifuged 10,000g, 5mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

DNA was quantified using two methods: NanoDrop1000 & Qubit 3.0 (ThermoFisher).

For the Qubit, the samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit Data (Google Sheet): 20151125_qubit_gDNA_geoduck_oly_quants

METHOD CONCENTRATION (ng/μL) TOTAL (μg)
Qubit 137 27.4
NanoDrop1000 295 59.0

 

Yield is solid. We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.

The NanoDrop & Qubit numbers still aren’t close (as expected).

The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.

 

NanoDrop Absorbance Values & Plots

 

 

DNA Isolation – Geoduck Ctenidia gDNA

Isolated additional gDNA for the genome sequencing. In an attempt to obtain better yields, I used ctenidia (instead of adductor muscle). Additionally, to try to improve the quality (260/280 & 260/230 ratios) of the gDNA, I added a chloroform step after the initial tissue homogenization.

Used 190mg of Panopea generosa ctenidia collected by Brent & Steven on 20150811.

  • Homogenized in 500μL of DNAzol.
  • Added additional 500μL of DNAzol.
  • Centrifuged 12,000g, 10mins, @ RT.
  • Split supernatant equally into two tubes.
  • Added 500μL of chloroform and mixed moderately fast by hand.
  • Centrifuged 12,000g, 10mins, RT.
  • Combined aqueous phases from both tubes in a clean tube.
  • Added 500μL of 100% EtOH and mixed by inversion.
  • Spooled precipitated gDNA and transferred to clean tube.
  • Performed 3 washes w/70% EtOH.
  • Dried pellet 3mins.
  • Resuspended in 200μL of Buffer EB (Qiagen).
  • Centrifuged 10,000g, 5mins, RT to pellet insoluble material.
  • Transferred supe to clean tube.

DNA was quantified using two methods: NanoDrop1000 & Qubit 3.0 (ThermoFisher).

For the Qubit, the samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit Data (Google Sheet): 20151125_qubit_gDNA_geoduck_oly_quants

METHOD CONCENTRATION (ng/μL) TOTAL (μg)
Qubit 105 21.0
NanoDrop1000 173 34.6

 

Yield is definitely much, much better than adductor muscle! Should’ve switched to a different tissue a long time ago! We should finally have sufficient quantities of gDNA to allow for BGI to proceed with the rest of the genome sequencing! Will run sample on gel to evaluate integrity and then send off to BGI.

The NanoDrop & Qubit numbers still aren’t close (as expected).

The addition of the chloroform step definitely helped improve the 260/280 OD ratio (see below). However, the addition of that step had no noticeable impact on the 260/230 OD ratios, which is a bit disappointing.

 

NanoDrop Absorbance Values & Plots

 

Phenol-Chloroform DNA Cleanup – Geoduck 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 (280μL) of chlforoform:isoamyl alcohol (24:1)
  • Mixed by hand – moderate shaking
  • Centrifuged 2mins, 10,000g, RT
  • Transferred aqueous phase (210μL) to clean tube
  • Added 0.1vols (21μL) of 3M sodium acetate (pH = 5.2)
  • Added 2vols (420μL) 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 371.83 100 37,183
Quant-IT 100.83 100 10,082

 

The NanoDrop1000 overestimates the concentration of the sample by 3.7x!

Regardless, the yield isn’t all that great (using yield from Quant-IT), which has generally been the case for all of the geoduck gDNA isolations I’ve performed. It would probably be prudent to try isolating gDNA from a different tissue to see if yields improve…

Will evaluate gDNA quality on a gel.

Fluorescence (Google Sheet): 20151124_geoduck_oly_gDNA_quants

 

NanoDrop1000 Measurements and Plots

The clean up procedure didn’t really seem to help with the geoduck sample, as we’re still seeing a significant amount of absorbance from 230 – 250nm.

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.