Samples Received – White Abalone DNA from CDFW Shellfish Health Lab

Received white abalone (Haliotis sorenseni) DNA extracted from digestive gland, post-esophagus, and feces from Jim Moore and Blythe Marshman at the California Dept. of Fish & Wildlife.

These are intended for qPCR to assess presence of the RLOv.

Samples were stored in the big -20C in FSH 240.

 

Sample ID – Black Abalone DNA for RLOv qPCRs

Carolyn & Stan Langevin have agreed that the DNA helicase qPCR should be tested on 10 black abalone DNA extractions that fall into multiple levels of the Friedman Lab’s withering syndrome histology scoring.

Downloaded the (Google Sheet) Black Abalone: Expt 1 – WS & Phage as a CSV file. After downloading, I renamed the file (Black_Abalone.csv) to facilitate easier usage in the following steps.

Created a sqlite database using GitBash for Windows:
Change to directory where file is located:

$cd Downloads

Start sqlite:

$sqlite3

Tell sqlite that the field separator will be commas (i.e. CSV file):

sqlite>.separator ","

Import the CSV file and provide a name for the resulting database:

sqlite>.import Black_Abalone.csv BlackAbs

Set output display mode to column for easier reading:

sqlite>.mode column

Set output display to include column headers:

sqlite>.headers on

 

To select all the samples that have scores of 0 in both PE and DG RLO fields (screen cap does not show entire output list):

 

To select all the samples that have scores of 1 in both PE and DG RLO fields:

 

To select all the samples that have scores of 2 in both PE and DG RLO fields:

 

Here are the full set of results in a table

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
06:5-03 06:5-35A 06:5-31
06:5-04 06:50-08 06:5-32B
06:5-08 06:50-10 06:6-46
06:5-09 06:6-32 06:6-49
06:5-10 06:6-39 08:3-05
06:5-11 06:6-42 08:3-07
06:5-14 06:6-44 08:3-15
06:5-16 06:6-52 08:3-16
06:5-18 06:6-54
06:5-20 07:12-18
06:5-21 08:3-08
06:5-22 08:3-10
06:5-24
06:5-30
06:50-04
06:50-05
06:50-11
06:50-12
06:50-13
06:50-15
06:50-16
06:6-01
06:6-02
06:6-03
06:6-05
06:6-08
06:6-11
06:6-12
06:6-13
06:6-15
06:6-16
06:6-17
06:6-18
06:6-20
06:6-21
06:6-22
06:6-23
06:6-24
06:6-25
06:6-26
06:6-27
06:6-28
07:12-01
07:12-02
07:12-03
07:12-04
07:12-05
07:12-06
07:12-07
07:12-09
07:12-10
07:12-13
07:12-19
08:3-01
08:3-02
08:3-03
08:3-04
08:3-13
08:4-01
08:4-02
08:4-03
08:4-04
08:4-05
08:4-06
08:4-07
08:4-08
08:4-09
08:4-10
08:4-11
08:4-12
08:4-13
08:4-14
08:4-15
08:4-16
08:4-17
08:4-18
08:4-19
08:4-20
08:4-21
08:4-22
08:4-23
08:4-24
08:4-25
08:5-06

Will select just 10 of those in the RLO/RLOv 0 column for use in qPCR.

I was able to track down the boxes where are these DNAs were stored (see images below).

Boxes that were not labeled with accession numbers of the samples contained therein are now labeled.

Boxes that contained samples that belonged in other boxers were transferred to the appropriate box.

All boxes were located, and returned, to the big -20C in 240 on Lisa’s shelf.

Samples for Abalone Withering Syndrome ddPCR

I selected the following samples (Ab Endo 2011 Water Filter DNA samples) to send to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR):

  1. CI SRI CP 1A (0 copies)
  2. CI SRI CP 2B (highest)
  3. CI SRI CP 2A (0 copies)
  4. CI SRI CP 1B (high)
  5. CARMEL +500M 1 (medium)
  6. CARMEL +500M 2 (low)

10μL of each sample was sent. Tubes were labelled with “DNA #”. The ‘#’ corresponds to the number in the list above.

10μL of each of p18RK7 qPCR standards (from 20120730) were sent.

Two sets of WSN1 F/R working stocks (10μM) were also sent.

Also sent the QX200 Droplet Generation Oil for EvaGreen Kit (BioRad) that we ordered.

Shipment was on “wet” ice.

qPCR – Evaluation of withering syndrome and phage presence in holding tanks

In anticipation of receiving a large quantity of abalone from Japan, Carolyn wants to assess  current infection status of our abalone to make decisions on how/where to house the incoming abalone.

Ran a qPCR to detect withering syndrome and the withering syndrome phage on the following DNA samples isolated today by Lisa:

  • RR1 (Haliotis discus discus) – Seawater DNA
  • RR2 (Haliotis diversicolor) – Seawater DNA
  • 14:5-1 – 4- Dg DNA

Positive control: pCR2.1/ORF25 (1:1000) from 20141008.

Primers used:

  • Withering Syndrome – WSN1F/R
  • Phage – 1_ORF25F_225_CSF, 1_ORF25R_399_CSF,

All samples were run in duplicate.

Master mix calcs are here: 20150316 – qPCR H.discus H2O and feces

Plate layout, cycling params, etc. can be viewed in the qPCR Report (see Results).

Results:
qPCR Report (PDF): Sam_2015-03-16 16-46-06_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-03-16 16-46-06_CC009827.pcrd

Withering syndrome
– standard curve is perfect
– all samples, except seawater RR2, amplified

Phage
– no standard curve; this is not ready yet; as such, you can ignore the copy number (SQ) listed in the data file
– consistent amplification in both seawater samples (RR1, RR2)
– zero or inconsistent (i.e. one of two reps amplified) in remaining samples
– melt curves in the RR1 and RR2 samples exhibit multiple peaks, suggesting amplification of multiple targets (i.e. lack specificity)
– melt curves in the remaining samples only exhibit single peaks
– RR2 melt curves are shifted 2C (82C), compared to all other samples (80C)

PCR – Ireland Clam RLO DNA S/6/14 #19 (from 20150130)

After previously confirming that the issue with previous PCRs was due to bad reagents, I re-ran the PCR on the clam RLO DNA isolated 20150130 using a set of universal 16s primers, as well as a universal 18s primer set to serve as a positive control that amplifiable DNA was present in the sample.

Master mix calcs are here: 20150219 – cPCR Universal Primers Apex Red MM

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

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 1mins

Samples were run on 1.0% agarose, low TAE gel stained w/EtBr.

Results:

Ladder used was O’GenRuler 100bp DNA Ladder (Thermo-Fisher).

No sample was loaded directly next to ladder to facilitate excision, if necessary.

Each sample was accompanied by a no template control (NTC).

The ehrlichia universal primers (EHR) and the universal 18s (18s) primers are the only two primer sets that do not have contamination present in the NTCs.

Excised the EHR band and purified with Ultrafree-DA columns (Millipore). Purified DNA was stored @ -20C and will be used for cloning/sequencing next week.

Have already ordered additional primer sets of those above that are contaminated. Will re-run the PCR with those new, sterile primer sets when they arrive to obtain a larger product (the EHR amplicon is only ~350bp).

DNA Quantification & PCR – Ireland Clam S/6/14 #19 DNA

Quantification

Quantified the DNA isolated 20150130 via NanoDrop1000 (Thermo Fisher) for quick assessment of DNA.

Although the NanoDrop1000 overestimates actual yields, this is still interesting because the overall yield of this sample is greater than either of the samples isolated on 20150122, yet had significantly less starting material.

 

PCR

Ran PCRs with the following “universal” primer sets in attempt to amplify a 16s (prokaryote) fragment from the RLO that is present in this sample. Additionally, ran a universal 18s (eukaryote) primer set to verify the presence of any amplifiable DNA in the sample, in case none of the 16s primers work.

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

Master mix calcs are here: 20150203 – cPCR Clam debris DNeasy

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 from yesterday’s PCR out on a 0.8% agarose,  1x TBE gel w/EtBr

Results:

Ladder is Hyperladder I (Bioline)

Well, ironically, the only thing that shows amplification is the no template controls (NTC) in the universal 16s primer set!  The only useful aspect of this is that it demonstrates that the reagents are functional.

The universal 18s primers don’t seem to amplify anything, either.

Tomorrow, I’ll test these primers out on DNA that I know will amplify, instead of these new clam DNA samples.

DNA Isolation – Ireland Clam Sample S/6/14 #19

Previous attempts at isolating usable DNA failed.  Previously used both the QIAamp Fast DNA Stool Kit and DNeasy Kit (Qiagen) and both yielded nothing. In a last ditch effort, since there’s no tissue left, I pellet the remaining tissue/debris, removed the EtOH and processed the sample with the DNeasy Kit (Qiagen), following the animal tissues spin protocol.  DNA was eluted with 100uL of Buffer AE.

Will quantify and PCR next week.

DNA Isolation – Ireland Clam Tissue

Performed DNA isolation on clam tissue (sample: S/6/14 #19) supplied by Deborah Cheslett in July 2014 (based on date on letter accompanying the sample).  Sample was preserved in 80% EtOH.  Isolated DNA using the QIAamp Fast DNA Stool Kit (Qiagen), per Carolyn’s request.

Removed tissue from EtOH, blotted dry with Kim Wipes.  Tissue type was not noted in the letter accompanying the sample.

Tissue weighed 117mg, which is just below the recommended range for the the Qiagen stool kit (180 – 220mg is recommended).  Minced tissue with razor blade and processed according to the manufacturer’s protocol for pathogen detection.  Because tissue was very dense/rubbery, the tissue did not lyse during the initial incubation period (95C for 5mins).  Extended this incubation to 2.5hrs in an attempt to lyse the tissue.  Lysis did not fully dissolve the tissue, which was not surprising.

Proceeded with the manufacturer’s protocol and eluted with 100μL of Buffer ATE.

Retained remaining supernatant from the lysis step.

Processed the remaining unlysed tissue using the DNeasy Blood & Tissue Kit (Qiagen) according the manufacturer’s protocol.  Tissue lysis step was performed at 56C O/N.

Sample was eluted with 200μL of Buffer AE.