Category Archives: Geoduck Genome Sequencing

Geoduck Genome Sequencing

Data Management – Illumina Geoduck HiSeq & MiSeq Data

The HDD we received from Illumina last week only had data (i.e. fastq files) from the NovaSeq runs they performed – nothing from either the MiSeq, nor the HiSeq runs.

We contacted them about the missing data, they confirmed it was missing, and uploaded the remaining data to BaseSpace.

Began downloading the data – will take awhile…

 

Files will be temporarily stored in these locations:

/volume1/web/nightingales/Geoduck_MiSeq/170317_M03814_0172_000000000-B2K79/Data/GeoDuckRNAMiSeq-35978947

/volume1/web/nightingales/Geoduck_HiSeq/170228_ST-K00104_0382_BHHGTLBBXX/Data/Ironman-35682656

/volume1/web/nightingales/Geoduck_HiSeq/170228_ST-K00104_0381_AHHHWNBBXX/Data/Ironman-35682656

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Data Received – Geoduck Genome Sequencing by Illumina

We previously sent some geoduck samples to Illumina, as part of a pilot project for them to test out a new sequencing platform. The data has finally arrived!

It was sent on a 4TB Seagate external hard drive.

Due to weird connection issues we’ve recently encountered with our server, Owl (Synology DS1812+), I connected the HDD directly to Owl via USB (instead of connecting to a computer and transferring). I transferred the data using the Synology web interface to avoid any computer/NAS connection issues that might interrupt the transfer.

We have a meeting with the Illumina people tomorrow afternoon to review the data they’ve provided (looks like it’s going to take awhile, though). Once that meeting takes place, we’ll figure out how to document this project in our data management plan.

 

 

 

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DNA Isolation – Geoduck gDNA for Illumina-initiated Sequencing Project

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.

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DNA Isolation – Geoduck gDNA for Potential Illumina-initiated Sequencing Project

We were approached by Cindy Lawley (Illumina Market Development) yesterday to see if we’d be able to participate in some product development. We agreed and need some geoduck DNA to send them, in case she’s able to get our species greenlighted for use.

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 two separate 50mg 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 = 19.4ng/μL (Quant data is here [Google Sheet]: 20161221_gDNA_qubit_quant

Yield is low (~1.8μg), but have enough to satisfy the minimum of 1μg requested by Cindy Lawley.

Evaluated gDNA quality (i.e. integrity) by running ~250ng (12.5μL) 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 sample looks good. Strong, high molecular weight band is present with minimal smearing. However, there is a smear in the ~500bp range. This is most likely residual RNA. This is surprsing since the E.Z.N.A Mollusc Kit includes n RNase step. Regardless, having intact, high molecular weight DNA is the important part for this project. Will prepare to send remainder (~1.5μg) of geoduck to Illumina with other requested samples.

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Data Management – Integrity Check of Final BGI Olympia Oyster & Geoduck Data

After completing the downloads of these files from BGI, I needed to verify that the downloaded copies matched the originals. Below is a Jupyter Notebook detailing how I verified file integrity via MD5 checksums. It also highlights the importance of doing this check when working with large sequencing files (or, just large files in general), as a few of them had mis-matching MD5 checksums!

Although the notebook is embedded below, it might be easier viewing via the notebook link (hosted on GitHub).

At the end of the day, I had to re-download some files, but all the MD5 checksums match and these data are ready for analysis:

Final Ostrea lurida genome files

Final Panopea generosa genome files

Jupyter Notebook: 20161214_docker_BGI_data_integrity_check.ipynb

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Data Management – Download Final BGI Genome & Assembly Files

We received info to download the final data and genome assembly files for geoduck and Olympia oyster from BGI.

In total, the downloads took a little over three days to complete!

The notebook detailing how the files were downloaded is below, but it should be noted that I had to strip the output cells because the output from the download command made the file too large to upload to GitHub, and the size of the notebook file would constantly crash the browser/computer that it was opened in. So, the notebook below is here for posterity.

Jupyter Notebook: 20161206_docker_BGI_genome_downloads.ipynb

 

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Data Management – Geoduck Small Insert Library Genome Assembly from BGI

Received another set of Panopea generosa genome assembly data from BGI back in May! I neglected to create MD5 checksums, as well as a readme file for this data set! Of course, I needed some of the info that the readme file should’ve had and it wasn’t there. So, here’s the skinny…

It’s data assembled from the small insert libraries they created for this project.

All data is stored here: http://owl.fish.washington.edu/P_generosa_genome_assemblies_BGI/20160512/

They’ve provided a Genome Survey (PDF) that has some info about the data they’ve assembled. In it, is the estimated genome size:

Geoduck genome size: 2972.9 Mb

Additionally, there’s a table breaking down the N50 distributions of scaffold and contig sizes.

Data management stuff was performed in a Jupyter (iPython) notebook; see below.

Jupyter Notebook: 20161025_Pgenerosa_Small_Library_Genome_Read_Counts.ipynb

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