Category Archives: Miscellaneous

Genome Assembly – Olympia oyster PacBio minimap/miniasm/racon

In this GitHub Issue, Steven had suggested I try out the minimap/miniasm/racon pipeline for assembling our Olympia oyster PacBio data.

I followed the pipeline described by this paper: http://matzlab.weebly.com/uploads/7/6/2/2/76229469/racon.pdf.

This notebook entry just contains the racon execution. This produced this assembly:

http://owl.fish.washington.edu/Athaliana/201709_oly_pacbio_assembly_minimap_asm_racon/20170918_oly_pacbio_racon1_consensus.fasta

All intermediate files generated from this pipeline are here:

http://owl.fish.washington.edu/Athaliana/201709_oly_pacbio_assembly_minimap_asm_racon/

I’ll put together a TL;DR post that provides an overview of the pipeline and an assessment of the final assembly.

Previously ran minimap
and then miniasm.

Jupyter Notebook (GitHub): 20170918_docker_pacbio_oly_racon0.5.0.ipynb

RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks

My previous go at this was a little premature – I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that Grace has gone through and annotated the blocks to point out gonad tissue. SN-10-16 was sent to Katherine Silliman on 20170720.

Isolated RNA from Olympia oyster gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Laura’s notebook for full details on samples and preservation.

 

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.

Background on all of this is in this GitHub Issue

NF-10-22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

IMPORTANT:

  • Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1)

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 20,000g.
  • Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
  • Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice.

 

Results:

Samples were not quantified due to lack of proper RNA Qubit assay AND the computer that our NanoDrop1000 is hooked up to is dead. Will have Katherine Silliman perform quantification.

Samples were stored at -80C temporarily.

Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.

RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks

UPDATE 20170712: The RNA I isolated below is from incorrect regions of tissue. I misunderstood exactly what this tissue was, and admittedly, jumped the gun. The tissue is actually collected from the visceral mass – which contains gonad (a small amount) and digestive gland (a large amount). The RNA isolated below will be stored in one of the Shellfish RNA boxes and I will isolate RNA from the correct regions indicated by Grace

Isolated RNA from Olympia oyster gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Laura’s notebook for full details on samples and preservation.

 

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.

Tissue identification is available in this GitHub Issue

NF-10-22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

IMPORTANT:

  • Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1).
  • Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the “-20C Kit Components” box.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 20,000g.
  • Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
  • Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab Qubit 3.0 with the RNA High Sensitivity Assay (ThermoFisher Scientific) using 5μL of each sample.

Results:

Concentrations (Google Sheet): 20170710_RNA_qubit_oly_histo_blocks

Well, the good news is that there’s RNA from all the samples and it seems to be in relatively high concentrations!

The bad news is that the concentrations for 10 of the 12 samples were too high and outside the range of the Qubit RNA HS Assay! Since we don’t have the broad range RNA assay, I can’t properly quantify the remaining samples. However, these samples are being sent to Katherine Silliman at some point, so I’ll leave it up to her to quantify the samples. I’m also guessing that she’ll run them on a Bioanalyzer to assess their integrity prior to beginning library construction, so that will also yield concentrations for the samples.

Samples were stored at -80C temporarily.

Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.

Sample Annotation – Olympia oyster histology blocks (from Laura Spencer)

I’ve been asked to isolate RNA from some paraffin-embedded Olympia oyster gonad tissue.

Despite some excellent documentation by Laura Spencer (images of tissue layouts in histology cassettes and a corresponding cassette mapping key file), the histology facility seems to have flipped some things around and/or repositioned/split the contents of each cassette. This makes ID-ing the proper tissues tedious and, at times, difficult.

The list of tissues that needs to be processed is listed in this GitHub Issue #648. I’ve also added the list below:

NF-10 22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

Prior to beginning RNA isolations, I have annotated images of the histology blocks and will be waiting for Laura to confirm that my annotations are correct. I will be posting a link to this notebook entry in the GitHub issue listed above for her to view and wait for her confirmation.

UPDATE 201700707 – Laura has indicated that many of my annotations are incorrect. Katie has gone through and made proper identification: https://github.com/sr320/LabDocs/issues/648#issuecomment-313792588

 

Additionally, as indicated in the GitHub Issue above, histology block “Oly 14″ does not have a corresponding tissue cassette photo (containing sample NF-10 26). Without the original image, I don’t think I can make an accurate guess on how the tissues are oriented in the resulting two histo blocks (see below).

 

BLOCKS 5

 

BLOCK 6

 

 

BLOCK 9

 

 

BLOCK 10

 

 

BLOCKS 14 (unable to annotate at time of posting)

 

 

 

BLOCK 15

 

 

 

BLOCK 21

 

 

 

 

BLOCK 22

Manuscript Re-submission – Oly Stress Response to PeerJ for Review

Last August, we made our initial submission of this paper to PeeJ.

Today, we re-submitted the revised manuscript.

The repo for this paper is here.

I’ve also submitted an updated pre-print. I will update this post when it is publicly accessible (it has to be approved by PeerJ staff before it becomes public).

UPDATE 20170703 – Updated pre-print is now available: https://peerj.com/preprints/1595/

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)

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).

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

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.

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