Tag Archives: Panopea generosa

Data Received – Initial Geoduck Genome Assembly from BGI

The initial assembly of the Ostrea lurida genome is available from BGI. Currently, we’ve stashed it here:

http://owl.fish.washington.edu/P_generosa_genome_assemblies_BGI/20160314/

The data provided consisted of the following three files:

  • md5.txt
  • N50.txt
  • scaffold.fa.fill

md5.txt – Checksum file to verify integrity of files after downloading.

N50.txt – Contains some very limited stats on scaffolds provided.

scaffold.fa.fill – A FASTA file of scaffolds. Since these are scaffolds (and NOT contigs!), there are many regions containing NNNNNN’s that have been put in place for scaffold assembly based on paired-end spatial information. As such, the N50 information is not as useful as it would be if these were contigs.

Additional assemblies will be provided at some point. I’ve emailed BGI about what we should expect from this initial assembly and what subsequent assemblies should look like.

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Data Received – Panopea generosa genome sequencing files from BGI

Downloaded data from the BGI project portal to our server, Owl, using the Synology Download Station. Although the BGI portal is aesthetically nice, it’s set up poorly for bulk downloads and took a few tries to download all of the files.

Data integrity was assessed and read counts for each file were generated. The files were moved to their permanent storage location on Owl: http://owl.fish.washington.edu/nightingales/P_generosa/

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: 1,208,635,950

BGI provided us with the raw data files for us to play around with, but they are also currently in the process of performing the genome assembly.

 

Jupyter Notebook file: 20160126_Olurida_BGI_data_handling.ipynb

Notebook Viewer: 20160126_Olurida_BGI_data_handling.ipynb

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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

 

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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.

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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

 

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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.

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DNA Isolation – Geoduck Adductor Muscle gDNA

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

Isolated gDNA from 257mg adductor muscle tissue collected by Steven & Brent on 20150811.

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 54.93 79 4,339
Quant-IT 34.52 79 2,727

 

The NanoDrop1000 overestimates the concentration of the sample by 1.6x!

Regardless, the yield isn’t all that great, 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

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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.

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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…

 

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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.

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