Author Archives: kubu4

GitHub Curation

Updated a couple of GitHub Wikis:

 


 

Created a new repo in the RobertsLab Organization GitHub account with a wiki to provide an overview of how to use of Hyak (mox) computing node. This was lightly modified from what Sean already had in his personal repo.

 


 

As a quick test, I updated all the md files in  the sr320/LabDocs/code md files to format headers for GitHub’s newest interpretation of headers. The headers (represented by a series of ‘#’) require a space between them and the subsequent text.  I used the following command in bash:

for i in *.md; do sed -i.bak 's/^#*/& /g' "$i"; done

The code works as follows:

  • Run for loop on all .md files in the directory

  • Use sed to edit the files in place: -i.bak (this command structure is needed for Mac OS X).

  • 's/^#*/& /g': Performs a substitution by identifying all lines beginning (^) with a pound symbol (#) and match zero or more occurrences of the pound symbol (*), then substituting the same pattern that was matched and adding a space at the end of the pattern (& ). Do this for all occurrences found within the document (g).

Since this worked, I’ll probably run this through all of the md files in all of our various repos to quickly and easily fix header formatting issues.

 


 

Working on updating the Genome-sequencing-December-2016-(UW-PacBio) wiki, but need to work out the kinks on any easy, documentable way to rename and move some files around in order to make files/organization compliant with our data management plan (DMP).

 

Current strategy:

  • Generate MD5 checksums for fastq files for each of the SMRT cell runs.

  • Copy file names from the the .xml file in the top level of each SMRT cell run folder to an array.

  • Use parameter substitution (in bash) to strip path and suffix from each index of the array (results likely stored in a secondary or tertiary array).

  • Use bash find command to copy the filtered_subreads.fastq.gz from each SMRT cell run folder, and append each of the corresponding stripped filenames in the final array to the beginning of the fastq file, to the owl/nightingales/O_lurida directory.

  • Generate new MD5 checksums on the copied files and compare to original MD5 checksums. This will confirm two things: 1 – The data did not get corrupted when copied. 2 – The new filenames correspond to the correct, original filtered_subreads.fastq.gz file (renaming a file doesn’t alter the MD5 checksum).

  • Archive the original SMRT cell run folders (which contain a ton of metdata files)

Share

qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on samples extracted earlier today. Also re-ran samples 15:08-29 and 15:09-145, per Ava’s request.

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170619_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

qPCR Report (PDF): Sam_2017-06-19 13-10-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-06-19 13-10-22_CC009827.pcrd

 

These will need to be re-run, as the standard curve is a bit wonky (see below). Will re-run later this week.

 

Share

DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 19 red abalone digestive gland tissue samples.

Tissue was weighed and then DNA extracted.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples were stored at 4C in FSH240 in racks for qPCR later today.

Sample information is in this spreadsheet (Google Sheet): Ava WS Transmission DNA Extractions

Share

Goals – June 2017

Well, my previous goal was to tidy up an existing manuscript and get it re-submitted to PeerJ. That’s pretty much done, as Steven will be giving a final once over and formatting the rebuttal letter prior to resubmission.

June will be a bit of a short month for me, due to some travel, but here’re some things on the agenda:

  • Update the Oly Genome Wiki to accurately reflect the most recent PacBio Sequencing we had done.

  • Related to the above goal is updating Nightingales to house just the raw sequencing data files for the Oly PacBio sequencing, while archiving the associated meta data (QC files, reports, etc).

  • Related to THAT goal is then updating our Nightingales spreadsheet to reflect, and provide links to, the raw sequencing files.

  • Establish (and build out) an “On Boarding” repo in the Roberts Lab GitHub Page. This should make it easier for new lab members to find the various resources they need. More importantly, it should make it easier for us to direct people to find that info!

Share

DNA Methylation Quantification – Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)

Used the MethylFlash Methylated DNA Quantification Kit (Colorimetric) from Epigentek to quantify methylation in these coral DNA samples.

All samples were run in duplicate <em>except</em> 2h Block 1 due to insufficient DNA.

The following samples were used in a 1:10 dilution (2uL DNA : 18uL NanoPure H2O), due to their relatively high concentrations, to ensure accurate pipetting:

  • 72h Block 4
  • D14 Block 1
  • D14 Block 2
  • D14 Block 3
  • D14 Block 4
  • D14 Block 5
  • D14 Block 6
  • D14 Block 8
  • D14 Block 10

All samples were diluted to a final concentration of 9.645ng/uL (154.24ng total; 17.6uL) in NanoPure water, which is equal to 77.12ng of DNA per assay replicate. These numbers were chosen based off of the sample with the lowest concentration.

The following samples were used in their entirety:

  • 2h Block 8
  • D35 Block 8

Calculations were added to the spreadsheet provided by Javier (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx

The spreadsheet became overly complicated because I initially forgot to account for the need to run each sample in duplicate.

The kit reagent dilutions were as follows:

  • Diluted ME1: 52mL of ME1 + 464mL of <em>distilled</em> water
  • Diluted ME4: 10uL of ME4 + 10uL of TE Buffer (pH=8.0; made by me on 20130408).
  • Standard curve: Prepped per instruction manual, with double volumes for two plates.
  • Diluted ME5: 50uL/well x 152well = 7600uL; 7600uL/1000 = 7.6uL; 7.6uL ME5 + 7592.4uL Diluted ME1
  • Diluted ME6: 50uL/well x 152well = 7600uL; 7600uL/2000 = 3.8uL; 3.8uL ME6 + 7596.2uL Diluted ME1
  • Diluted ME7: 50uL/well x 152well = 7600uL; 7600uL/5000 = 1.52uL; 1.52uL ME7 + 7598.48uL Diluted ME1

All diluted solutions were stored on ice for duration of procedure.

The remaining Diluted ME1 solution was stored at 4C (FTR 209), and is stable for 6 months, per the manufacturer’s instructions.

See the Results section below for plate layouts.

Plates were read at 450nm on the Seeb Lab Victor 1420 Plate Reader (Perkin Elmer) and the amount of DNA methylation was determined.

Results:

Individual sample methylation quantification (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx

Plate Reader Output File Plate #1 (Google Sheet): 20170511_coral_DNA_methylation_plate01.xls

Plate Reader Output File Plate #2 (Google Sheet): 20170511_coral_DNA_methylation_plate02.xls

 

I’m not familiar with the experimental design, so I’m not going to spend time handling any of the in-depth analysis at this point in time. However, here’s the background on how methylation quantification and percent methylation were determined.

  1. Mean absorbance (450nm) was determined for all samples and standard curve samples. It’s important to note that the standard deviation between replicates was not evaluated and there appears to be consistent variability between samples, but I’m not certain how much variation is “acceptable” with and assay of this nature.

  2. The mean absorbance of the standard curve samples were plotted against their corresponding DNA amounts and a linear trendline was fitted to the points.

  3. Per the manufacturer’s recommendations, the four points (including the zero point) that yielded the best linear fit (i.e. best R^2 value) were used and the slope of best fit line for those four points was determined.

  4. This slope was then utilized in the equation provided by the manufacturer (see pg. 8 of the MethylFlash Kit manual).

Share

DNA Quantification – Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)

I quantified the three samples (listed below) that I SpeedVac’d yesterday using the the Roberts Lab Qubit 3.0.

  • 2h Block 1
  • 2h Block 8
  • D35 Block 8

Quantification was performed using the dsDNA Broad Range Kit.

Used 1uL of each sample.

Results:

One sample (2h Block 1) is still slightly too dilute in order to use the recommended total amount of DNA for the methylation assay (100ng), but still falls well within the recommended range for the assay. Will proceed with the methylation assay for all samples.

Values were added to the spreadsheet provided by Javier (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx

 

Qubit output file (Google Sheet): 20170511_qubit_A_cervicornis_DNA

Share

DNA Concentration – Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)

Three samples (of the 62 total) that were quantified earlier today, had concentrations too low for use in the methylation assay:

  • 2h Block 1
  • 2h Block 8
  • D35 Block 8

These samples were dried to completion in a SpeedVac.

They will be allowed to rehydrate O/N in 10uL of Buffer EB (Qiagen) and will be re-quantified tomorrow morning.

Share

DNA Quantification – Acropora cervicornis (Staghorn coral) DNA from Javier Casariego (FIU)

DNA samples received yesterday were quantified using the Roberts Lab Qubit 3.0 to improve quantification accuracy (samples provided by Javier were quantified via NanoDrop, which generally overestimates DNA concentration) prior to performing methylation assessment.

Quantification was performed using the dsDNA Broad Range Kit.

Used 1uL of each sample.

Results:

Three samples are too dilute for immediate use in the MethylFlash Methylated DNA Quantification Kit (Colorimetric) – max sample volume is 8uL. Will have to concentrate them (will likely use SpeedVac to prevent sample loss).

Values were added to the spreadsheet provided by Javier (Google Sheet): A.cervicornis_DNA_Extractions(May_2017).xlsx

Qubit output file (Google Sheet): 20170510_qubit_A_cervicornis_DNA

 

Share

qPCR – Ava’s RLO Transmission Samples

Ran qPCRs on the DNA I extracted on 20170504 and earlier today.

The full list of samples is here (Google Sheet): 20170502_Ava_Ab_List

Standard curve was p18RK7 from 20161128.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20170509_qPCR_WSN1_Ava_Samples

Plate layouts, cycling params, etc. can be seen in the corresponding qPCR Reports (see Results below).

Baseline threshold was manually set to 580, based on Lisa’s development of the withering syndrome qPCR assay.

Results:

Curves look good on all runs (except the one that’s been noted and has been repeated). Will pass along to Ava and Carolyn.

qPCR Report (PDF): Sam_2017-05-09 07-29-36_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 07-29-36_CC009827.pcrd

 


This plate has a bad curve and needs to be re-run! It has been repeated below!

I’ve included this for posterity only!

qPCR Report (PDF): Sam_2017-05-09 08-56-22_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 08-56-22_CC009827.pcrd


 

 

qPCR Report (PDF): Sam_2017-05-09 10-21-15_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 10-21-15_CC009827.pcrd

qPCR Report (PDF): Sam_2017-05-09 11-44-42_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 11-44-42_CC009827.pcrd

 

qPCR Report (PDF): Sam_2017-05-09 13-07-46_CC009827.pdf
qPCR Data File (CFX96): Sam_2017-05-09 13-07-46_CC009827.pcrd

Share

DNA Quantification – Ava’s RLO Transmission DNA

Quantified the DNA I isolated on 20170504 and earlier today using the Roberts Lab’s Qubit 3.0 and the dsDNA Broad Range assay.

Used 1uL of each sample.

Results:

The following samples were below the level of sensitivity of the Qubit assay:

  • 15:09-142
  • 15:11-113
  • 15:11-147
  • 15:11-149

Qubit output data (Google Sheet): 20170509_Ava_RLO_quantification_qubit

An easier-to-read summary of all the samples is here (Google Sheet): 20170502_Ava_Ab_List

 

Share