# Computing – Owl Partially Restored

Heard back from Synology and they indicated I should click the “Repair” option to fix the System Partition Failed error message seen previously.

I did that and our data is now accessible again. However, all the user account info, scheduled tasks (e.g. Glacier backups, notebook backup script), IP configurations, mail configurations, etc. have all been reset.

I downloaded/installed the various packages needed to have the server accessible via the web and configured the IP address settings.

Have a note out to Synology to see if the configurations can be restored somehow. Once I hear back, we’ll get user accounts re-established.

Below is a chronological set of screen caps of the repair process:

Our data is still here! This is before performing the “Repair” operation, btw. It seems it just required some time to re-populate directory structure.

Still getting a “degraded” error message, but all drives appear normal. However, Disk 3 in the DX513 is not showing; possible cause for “degraded” status?

Set up manual IP settings by expanding the “LAN 1″ connection.

# Manuscript Writing – The “Nuances” of Using Authorea

I’m currently trying to write a manuscript covering our genotype-by-sequencing data for the Olympia oyster using the Authorea.com platform and am encountering some issues that are a bit frustrating. Here’s what’s happening (and the ways I’ve managed to get around the problems).

PROBLEM: Authorea spits out a browser-crashing “unresponsive script” message (actually, lots and lots of them; clicking “Stop script” or “Continue” just results in additional messages) in Firefox (haven’t tried any other browsers). This renders the browser inoperable and I have to force quit. It doesn’t happen all of the time, so it’s hard to pinpoint what triggers this.

SOLUTION: Edit documents in Git/GitHub. I have my Authorea manuscript linked to a GitHub repo, which allows me to write without using Authorea.com. This is how I’ll be doing my writing the majority of the time anyway, but I would like to use Authorea.com to insert and manage citations…

PROBLEM: Authorea remains in a perpetual “saving…” state after inserting a citation. It also renders the page strangely, with HTML <br></br> tags (see the “Methods” section in the screen cap below).

SOLUTION: Type additional text somewhere, anywhere. This is an OK solution, but is particularly annoying if I just want to go through and add citations and have no intentions of doing any writing.

PROBLEM: Multi-author citations don’t get formatted with “et al.” By default, Authorea inserts all citations using the following LaTeX format:

cite{Elshire_2011}

Result: (Elshire 2011).

This is a problem because this reference has multiple authors and should be written as: (Elshire et al., 2011).

SOLUTION: Change citation format to:

citep{Elshire_2011}

Other citation formatting options can be found here (including multiple citations within one set of parentheses, and referring in-text author name with only publication year in parentheses):

How to add and manage citations and references in Authorea

PROBLEM: When a citation no longer exists in the manuscript, it still persists in the bibliography.

SOLUTION: A known bug with no current solution. Currently, have to delete them from the bibliography by hand (or, maybe figure out a way to do it programatically)…

PROBLEM: Cannot click-and-drag some references from Mendeley (haven’t tested other reference managers) without getting an error. To my knowledge, the BibTeX is valid, as it appears to be the same formatting as other references that can be inserted via the click-and-drag method. There are some references it won’t work for…

SOLUTION: Use the search bar in the citation insertion dialogue box. Not as convenient and slows down the workflow for citation insertion, but it works…

After the failure of the prep-scale PCR for the RAD library construction, Katherin Silliman pointed out a potential problem (too much dNTPs). This was odd because I was following the Meyer Protocol and I used what was indicated.

Oddly, it turns out that Katherine’s version of the Meyer Lab 2bRAD protocol differed from what I had download. To add to the confusion, both protocols have the same file name. Here’s what I’m talking about:

The file on the left is the one I was using and the one on the right is the file Katherine is using (NOTE: The file name’s aren’t exact because they were saved to the same directory and the numbers in the parentheses were appended to the file name automatically)

I’ve updated our copy of the protocol in our GitHub account. However, Katherine informed me that she’s just been pulling up the Meyer Lab page to reference the protocol. So, it’s possible they made a change to the file after I initially downloaded it, but the change wasn’t indicated in the file name.

http://people.oregonstate.edu/~meyere/docs/

However, when discussing with Katherine, she made a good point and said she just scaled up the test-scale PCR. Since the test-scale PCR was successful, she didn’t see a need to make any changes.

Will try this procedure again with the correct protocol; probably by scaling up the test-scale PCR.

# Opticon2 Calibration

Jake and Steven recently noticed localized “hot spots” on most of Jake’s recent qPCRs, where higher levels of fluorescence were consistently showing up in interior portions of the plates than the outer portion of the plates.

Ordered 5nmol of 6-FAM T10 Calibration Standard from Biosearch Technologies and resuspended it in 50μL of 1x dilution buffer (10mM Tris-HCl pH8.0, 50mM NaCl, 5mM MgCl2) to make a 100μM solution. Buffer and dye were stored @ -20C after use.

Buffer calculations: Total Volume = 15mL

• 1.5mL of 100mM Tris-HCl
• 150μL of 5M NaCl
• 750μL of 100mM MgCl2

Made a working dilution of the 6-FAM dye of 300nM in 5mL of 1x dilution buffer (15uL of 100uM dye in 5mL of buffer).

Ran the calibration protocol on the Opticon2 (BioRad) using 50μL of dye in all wells when required by the calibration protocol.

Results:

### EMPTY PLATE MEASUREMENTS

Empty Plate – Channel 1 voltage measurements

Empty Plate – Channel 2 voltage measurements

Empty Plate – Ratio of Channel 1 to Channel 2 voltage measurements.

The empty plate measurements above show the expected low voltage measurements, but also show a  ~5-fold difference in min/max voltages in each channel. Additionally, the voltage ratios (the third image above) show a wavy pattern, but a smooth, even level from well-to-well is what would be expected if the Opticon was in measuring things properly.

DYE PLATE MEASUREMENTS

Dye Measurements – Channel 1 voltage measurements

Dye Measurements – Channel 2 voltage measurements

Dye Measurements – Channel 1 to Channel 2 voltage measurement ratios.

The voltages measured in each channel show the expected increase in voltages relative to the empty plate (> 10x voltage than empty plate). However, the spread between the min/max voltages in both channels is ~4-fold. Additionally, the ratio between the two channels still shows the wavy pattern across all the wells instead of the expected even ratio from well-to-well that should result from the calibration.

It appears the calibration has not resolved the issue.

To verify that calibration has failed, I ran two sets of qPCR “protocols” that simply read the dye plate to measure fluorescence across the plate in two plate orientations.

Dye Fluorescence – Original Orientation

Dye Fluorescence – Original Orientation with Fluorescence Graph

Dye Fluorescence – 180 Degree Rotation

Dye Fluorescence – 180 Degree Rotation with Fluorescence Graph

First thing to notice is that there’s clearly uneven fluorescence detection across the plate. Viewing the images that also contain the fluorescence graphs reveals a spread of ~8-fold between the highest and lowest fluorescence detection.

The second thing to notice is that, despite rotating the plate 180 degrees, the rotation has no effect on the fluorescence detected in each block location.

Both of these taken together provide strong evidence that there’s an issue with the machine.

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

In another troubleshooting attempt for this problematic BS-seq Illumina data, I’m going to use Trimmomatic to remove the first 39 bases of each read. This is due to the fact that even after the previous quality trimming with Trimmomatic, the first 39 bases still showed inconsistent quality:

Ran Trimmomatic on just a single data set to try things out: 2212_lane2_CTTGTA_L002_R1_001.fastq.gz

Notebook Viewer: 20150506_Cgigas_larvae_OA_trimmomatic_FASTQC

Jupyter (IPython) notebook: 20150506_Cgigas_larvae_OA_trimmomatic_FASTQC.ipynb

Results:

Trimmed FASTQ: 20150506_trimmed_2212_lane2_CTTGTA_L002_R1_001.fastq.gz

You can see how flat the newly trimmed data is (which is what one would expect).

Steven will take this trimmed dataset and try additional mapping with it to see if removal of the first 39 bases will improve the mapping.

# PCR – Test Universal Primers with Abalone DNA

Since I’ve had no success in amplifying any of the Ireland Clam RLO (S/6/14 #19) DNA, I’m testing all the universal primer sets I’ve previously tried on the Ireland Clam DNA with red abalone DNA known to have heavy withering syndrome infection (confirmed via histology and qPCR) to verify that these universal primer sets actually work.  I’m also using the withering syndrome primer sets on this DNA to function as a positive control.

Template DNA is: 09:20-08 (from tissue)

Background info for template DNA is here: Red/Pink/Pinto

Primers being used are:

• 16s/23s-F/R
• 27F, 1492R
• EHR16D, EHR16R (universal ehrlichia)
• EUB-A/B
• 18s EUK 581 F, 18s EUK 1134 R
• WSN1 (withering syndrome)

Master mix calcs are here: 20150204 – Ireland Clam Troubleshooting GoTaq Flexi

All samples were run in duplicate.

Cycling params were:
1 cycle of:

• 95C – 10mins

40 cycles of:

• 95C – 15s
• 50C – 15s
• 72C – 2mins

Ran samples out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

Nothing.  Since there’s nothing, I didn’t bother labelling the gel. So, this suggests that the PCR reactions aren’t working.  Will get newer reagents to replace the 5yr+ old reagents I have been using.  Also will try a different thermal cycler, just to rule out all possibilities.

# qPCR – p18RK7 Curve IDT Primer Test

Quick withering syndrome qPCR assay test using the ABI RLP_p probe and IDT primers. This is to see if we can still use IDT primers that have NOT been ABI HPLC purified. This would be a significant cost savings, as HPLC purified primers from ABI cost ~$60 each (“regular” IDT primers only cost ~$6 each). This should work.

Master mix calcs are here: 20140502 – qPCR p18RK7 Curve WSN1 IDT Primers

Used p18RK7 curve from 20120730 because we are running low on the p16RK7 curve (from the same date).

Plate layout, cycling params, etc can be found in the qPCR Report in the Results below.

Results:

qPCR Report (PDF): Sam_2014-05-02 09-42-38_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-05-02 09-42-38_CC009827.pcrd

Not surprising,the IDT primers work just fine. Will continue to use/order IDT primers for withering syndrome PCRs.

# RNA Clean Up – Colleen’s Sea Star Coelomycete RNA from 20140416

Zymoresearch support suggested putting the samples through another set of columns to help clean up the apparent phenol carryover that was seen (absorbance peak shifted to 270nm) in the initial isolation of these samples.

Added 500uL of TriReagent to each sample and vortexed. Then, proceeded with the remainder of the protocol (excluding the DNase step). Eluted with 50uL of 0.1% DEPC-treated H2O and spec’d on NanoDrop1000.

Results:

Absolutely horrible!! I can’t even begin to fathom what has happened here. The samples run with the sample kit all worked so well; why did this whole thing have to be jacked up with the actual samples??!!

Well, I’ll do a second elution using 50uL of 0.1%DEPC-treated H2O and spec. Let’s see if that helps….

OK, I didn’t even bother spec-ing all the samples because I noticed that the elution tubes had pellets in them! When I mix the tube prior to spec-ing (which is my normal behavior), I get the top absorbance spectra that is virtually useless. When I don’t mix the samples (thus, not disturbing the pellet), I get a more “realistic” spectra, but I can’t tell if I can trust it or not. I have contacted Zymoresearch support for more help with this…

It’s tempting to simply proceed with an EtOH precipitation, but I’m a bit concerned that the pellet in the tubes is resin from the column and that it might still bind some of the RNA. However, I guess the pellet is already in the elution solution, so the RNA should be soluble and, theoretically, not be able to bind to any residual resin…

# DNA Isolation – Test Sample

Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I’ve experienced.

Isolated gDNA from a C.gigas female gonad sample (EV2 141 go) provided by Mac. Isolated gDNA using DNazol (Molecular Research Center):

1. Incubated ~25mg of tissue O/N @ RT in 500uL of DNazol + 100ug/mL Proteniase K (2.7uL of 18.5mg/mL Fermentas stock) on rotator.

2. Added additional 500uL of DNazol and briefly disrupted remaining tissue with a few pipette strokes.

3. Pelleted debris by spinning 10mins, 10,000g @ RT.

4. Transferred supe to new tube and repeated Steps 3 & 4 one time.

5. Added 500uL of 100% EtOH; mixed by inversion.

NOTE: Despite initial appearance of white cloudy appearance after EtOH addition, cloudiness dispersed upon inversion and no visible DNA strands were present

1. Pelleted DNA by spinning 5000g 5mins @ RT.
2. Removed supe and washed pellet with 1mL of a 70% DNazol+30% EtOH solution.

3. Removed supe and washed pellet with 1mL 70% EtOH.

4. Repeated Step 8 two times.

5. Discarded supe, quick spun tube to pool residual EtOH. Removed all residual EtOH.

6. Resuspended in 200uL of TE (pH = 8.0) and incubated at RT for 5mins.

7. Pelleted insoluble material 12,000g 10mins @ RT.

8. Transferred supe to clean tube.

9. Spec’d on NanoDrop1000.

10. Ran ~500ng on 1.0% agaroase 1x modified TAE gel to evaluate integrity.

Results:

260/280 value looks excellent, but, as always seems to be the case with DNazol/TriReagent, the 260/230 value looks crappy. Will investigate gDNA integrity on agarose gel.

Lane 1 – Hyperladder I (Bioline)

Lane 2 – EV2 141 go C.gigas female gonad gDNA

Well, look at that! A nice, clear, high molecular weight band! It looks like my Buffer EB and/or nuclease-free water are is contaminated. Have discarded both. Will re-isolated Claire and Mac’s gDNA.

# Phenol-Chloroform DNA Clean Up – Mac and Claire’s Samples (from 20140410)

Due to low 260/230 values and Mac’s smeary sample, performed a phenol-chloroform DNA cleanup on the samples isolated 20140410.

1. Brought volume of each sample to 200uL with Buffer EB (Qiagen).

2. Added an equal volume (200uL) of 25:24:1 Phenol/Chloroform:Isoamyl alcohol.

3. Mixed on rotator for 20mins @ RT.

4. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

5. Transferred aqueous phase to new tube. Repeated steps 2-4 until samples exhibited no more interphase. Combined aqueous phases in to a single tube for each of the two samples.

6. Added and equal volume of chloroform (170uL).

7. Mixed on rotator for 20mins @ RT.

8. Separated aqueous/organic phases by spinning at 12,000g 5mins @ RT.

9. Transferred aqueous phase to new tube.

Performed an ethanol precipitation on each sample.

1. Added 0.1 volumes of 5M sodium acetate (pH = 5.2).
2. Added 2 volumes of ice cold 100% EtOH.

3. Incubated 20mins @ -20C.

4. Pelleted DNA by spinning 16,000g, 20mins @ 4C.

5. Discarded supe and washed pellets with 1mL 70% EtOH.

6. Pelleted DNA by spinning 16,000g, 5mins @ 4C.

7. Repeated steps 5-6 one time.

8. Removed all supernatant and resuspended in 100uL of nuclease-free H2O.

9. Spec’d on NanoDrop1000.

NOTE: Mac’s sample exhibited the same chunky/cloudiness upon addition of 100% EtOH that has been seen previously by both her and myself…

Results:

So, the clean up seemed to work wonders on the 260/230 values. Not surprisingly, Mac’s sample didn’t clean up nearly as nicely as Claire’s, based on my observations of the odd behavior during EtOH precipitation.

And, despite the nice, clean looking peaks, the 260/280 ratios are actually WORSE than the original isolation. Will run on gel for a further assessment of quality/integrity.

Loaded 5uL of each sample (~600ng) on a 1.0% agarose, 1x modified TAE gel stained with ethidium bromide.

Gel Layout:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – Claire’s CgF gonad sample

Lane 3 – Mac’s gonad sample

Used Hyperladder I this time, which has a high molecular weight band of 10kb and a low molecular weight band of 200bp.

Well, this totally sucks. Both samples appear to consist of nothing but 150-200bp fragments. Is something actually degrading these samples? The Buffer EB I used during the initial extraction is certainly old. Possible source of degradation? Ugh. Maybe I’ll try this again, but resuspend in TE…