Tag Archives: Crassostrea gigas

Agarose Gel – Oly gDNA for BS-seq Libraries, Take Two

The gel I ran earlier today looked real rough, due to the fact that I didn’t bother to equalize loading quantities of samples (I just loaded 1μL of all samples regardless of concentration). So, I’m repeating it using 100ng of DNA from all samples.

Additionally, this gel also includes C.gigas samples that Katie Lotterhos sent to us to see how they look.

Ran a 0.8% agarose, low-TAE gel, stained with ethidium bromide.

Results:

 

Look at that! The samples look MUCH nicer when they’re not overloaded and uniformly loaded!

Most have a prominent high molecular weight band (the band that’s closes to the top of the ladder, not the DNA visible in the wells). All exhibit smearing, but 2NF1 shows a weird accumulation of low molecular weight DNA.

Katie’s C.gigas samples (M1, M2, M3) look similar to the Olympia oyster gDNA, however her samples appear to have residual RNA in them (the fuzzy band ~500bp).

Will discuss with Steven which samples he wants to use for bisulfite treament and library construction.

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Samples Received – C.gigas Tissue & DNA from Katie Lotterhos

Received 6 samples from Katie today. The box was labeled and stored @ -20C.

 

Here description of the samples, via email:

Lotterhos samples (gigas) arriving tomorrow

1) Mantle tissue samples of C. gigas were collected on 20140705 (source: Pipestem Inlet) by KEL
2) Extraction on 20141028 by VG using Qiagen DNAeasy Blood and Tissue Kit
3) Beadwash on 20150720 by VG using homemade sera-mag speed beads
4) Qubit 3.0 quantification on 20151206 by KEL and the following amounts were sent:

M1: 13 uL of 386 ug/mL
M2: 13.8 uL of 326 ug/mL
M3: 13.15 uL of 380 ug/mL (solution looked cloudy)

 

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Here’s a pic:

Oyster food!

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Here’re some pics:

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Here’s a pic:

Collecting & cleaning larvae on nylon screen.

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Larval Care – Pacific oyster larvae at PSRF Manchester

It’s Saturday. Yep, Saturday…

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Here’s a pic:

Saturday morning at the hatchery.

 

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping.

Prepared a spreadsheet for Dan to use to calculate necessary quantities of algae (two species) to achieve target concentrations:

Google Sheet: AlgaeFeedingCalcs

Here’re some pics of the day:

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Larval Care – Pacific oyster larvae at PSRF Manchester

Continued helping on Dan’s Pacific oyster familial crosses project at the Puget Sound Restoration Fund (PSRF) hatchery at Manchester Research Station in Manchester, WA. Pacific oyster (Crassostrea gigas) families were crossed based on earlier genotyping. Here’re some pics of the day:

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Bioinformatics – Trimmomatic/FASTQC on C.gigas Larvae OA NGS Data

Previously trimmed the first 39 bases of sequence from reads from the BS-Seq data in an attempt to improve our ability to map the reads back to the C.gigas genome. However, Mac (and Steven) noticed that the last ~10 bases of all the reads showed a steady increase in the %G, suggesting some sort of bias (maybe adaptor??):

Although I didn’t mention this previously, the figure above also shows an odd “waves” pattern that repeats in all bases except for G. Not sure what to think of that…

Quick summary of actions taken (specifics are available in Jupyter notebook below):

  • Trim first 39 bases from all reads in all raw sequencing files.
  • Trim last 10 bases from all reads in raw sequencing files
  • Concatenate the two sets of reads (400ppm and 1000ppm treatments) into single FASTQ files for Steven to work with.

Raw sequencing files:

Notebook Viewer: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC

Jupyter (IPython) notebook: 20150521_Cgigas_larvae_OA_Trimmomatic_FASTQC.ipynb

 

 

Output files

Trimmed, concatenated FASTQ files
20150521_trimmed_2212_lane2_400ppm_GCCAAT.fastq.gz
20150521_trimmed_2212_lane2_1000ppm_CTTGTA.fastq.gz

 

FASTQC files
20150521_trimmed_2212_lane2_400ppm_GCCAAT_fastqc.html
20150521_trimmed_2212_lane2_400ppm_GCCAAT_fastqc.zip

20150521_trimmed_2212_lane2_1000ppm_CTTGTA_fastqc.html
20150521_trimmed_2212_lane2_1000ppm_CTTGTA_fastqc.zip

 

Example of FASTQC analysis pre-trim:

 

 

Example FASTQC post-trim (from 400ppm data):

 

Trimming has removed the intended bad stuff (inconsistent sequence in the first 39 bases and rise in %G in the last 10 bases). Sequences are ready for further analysis for Steven.

However, we still see the “waves” pattern with the T, A and C. Additionally, we still don’t know what caused the weird inconsistencies, nor what sequence is contained therein that might be leading to that. Will contact the sequencing facility to see if they have any insight.

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