Category Archives: Geoduck Genome Sequencing

Geoduck Genome Sequencing

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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SRA Submission – Genome sequencing of the Pacific geoduck (Panopea generosa)

Adding our geoduck genome sequencing (sequencing done by BGI) to the NCBI Sequence Read Archive (SRS). The current status can be seen in the screen cap below. Release date is set for a year from now, but will likely bump it up. Need Steven to review the details of the submission (BioProject, Experiment descriptions, etc.) before I initiate the public release. Will update this post with the SRA number once we receive it.

Here’s the list of files uploaded to the SRA:

151114_I191_FCH3Y35BCXX_L1_wHAIPI023989-79_1.fq.gz
151114_I191_FCH3Y35BCXX_L1_wHAIPI023989-79_2.fq.gz
151122_I136_FCH3L2FBBXX_L7_wHAXPI023990-97_1.fq.gz
151122_I136_FCH3L2FBBXX_L7_wHAXPI023990-97_2.fq.gz
160103_I137_FCH3V5YBBXX_L3_WHPANwalDDAADWAAPEI-101_1.fq.gz
160103_I137_FCH3V5YBBXX_L3_WHPANwalDDAADWAAPEI-101_2.fq.gz
160103_I137_FCH3V5YBBXX_L4_WHPANwalDDAADWAAPEI-101_1.fq.gz
160103_I137_FCH3V5YBBXX_L4_WHPANwalDDAADWAAPEI-101_2.fq.gz
160103_I137_FCH3V5YBBXX_L5_WHPANwalDDABDLAAPEI-100_1.fq.gz
160103_I137_FCH3V5YBBXX_L5_WHPANwalDDABDLAAPEI-100_2.fq.gz
160103_I137_FCH3V5YBBXX_L5_WHPANwalDDACDTAAPEI-102_1.fq.gz
160103_I137_FCH3V5YBBXX_L5_WHPANwalDDACDTAAPEI-102_2.fq.gz
160103_I137_FCH3V5YBBXX_L6_WHPANwalDDABDLAAPEI-100_1.fq.gz
160103_I137_FCH3V5YBBXX_L6_WHPANwalDDABDLAAPEI-100_2.fq.gz
151114_I191_FCH3Y35BCXX_L2_wHAMPI023988-81_1.fq.gz
151114_I191_FCH3Y35BCXX_L2_wHAMPI023988-81_2.fq.gz
160103_I137_FCH3V5YBBXX_L6_WHPANwalDDACDTAAPEI-102_1.fq.gz
160103_I137_FCH3V5YBBXX_L6_WHPANwalDDACDTAAPEI-102_2.fq.gz

Mate pair sequencing files were uploaded together within a single “Run”.

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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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