Tag Archives: phage

qPCR – CDFW White Abalone Samples (RLOv DNA helicase)

The samples that CDFW sent us earlier were previously checked for RLO presence with the withering syndrome qPCR assay.

Standard curve was from 20151106.

All samples were run in duplicate.

Master mix calcs are here; since I ran these with the other samples, the master mix used was part of the other project indicated in the spreadsheet (Google Sheet): 20170420 – qPCR RLOv DNA Helicase

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

Baseline threshold was manually set to 580.5, as previously determined.

Results:

qPCR Report (PDF): Sam_2017-04-20 07-50-18_CC009827.pdf
qPCR Data File (CFX): Sam_2017-04-20 07-50-18_CC009827.pcrd

Standard curve looks good and all samples provided come up positive for RLOv DNA helicase.

I’ve compiled the raw data of both the WSN qPCR and this in this Google Sheet: 20170420_CDFW_White_Ab_qPCR_summary

Here’s a summary table of the results (copy numbers are mean copies from qPCR replicates):

SAMPLE RLOV DNA HELICASE (COPIES) WSN1 (COPIES)
SF16-76_DG-1  165318.58 169.25
 SF16-76_DG-2  47839.81  20.70
 SF16-76_PE-1  1036697.17 633.75
 SF16-76_PE-2  46763.60  296.83
 SF17-17  117.29  2.16

NOTE: The WSN1 copies for SF17-17 is below the accepted detection limit of the qPCR assay (i.e. < 3 copies).

Will share my notebooks and spreadsheet with Blythe at CDFW.

Amplification Plots

Green = Standard Curve

Blue = Samples

Red = No template control

 

 

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qPCR – Black Abalone with XC Prophage Portal Primers

Ran qPCR with black abalone samples from the 1st and 2nd experiments to see if the Xenocal prophage portal gene is detected.

Master mix calcs (Google Sheet): 20160421 – qPCR Black Abs XenoCal phage portal

All samples were run in duplicate.

Black abalone sample 08:13-2 was run as a positive control.

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

Results:
qPCR Report (PDF): Sam_2016-04-21 14-11-09_CC009827.pdf
qPCR Data File (CFX): Sam_2016-04-21 14-11-09_CC009827.pcrd

Two samples failed to produce amplification: 06:6-44 and 07:12-18. All other samples amplified. Will compile this data with WSN and RLOv DNA helicase and send along to Carolyn and Stan.

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qPCR – RLOv DNA Helicase 2011 Water Filter DNA

Since we have a working qPCR for detecting the withering syndrome bacteriophage (RLOv), Carolyn wanted to see how detection/quantification compared to withering syndrome detection/quantification on water samples collected from various farms and their nearest wild abalone site.

DNA samples used were extractions from water filters collected for the Ab Endo Project in 2011.

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151228 – qPCR RLOv 2011 H2O Filters

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

qPCR LABEL FULL DESCRIPTION FARM/WILD SITE NEAREST FARM/WILD SITE
CI_SCI_PC_1A Painted Cave SCI 1A Wild The Cultured Abalone
CI_SRI_PC_1B Painted Cave SCI 1B Wild The Cultured Abalone
CI_SCI_PC_2B Painted Cave SCI 2B Wild The Cultured Abalone
CI_SCI_PC_2A Painted Cave SCI 2A Wild The Cultured Abalone
CI_SCI_PRIS_1A Prisoner’s 1A Wild The Cultured Abalone
CI_SCI_PRIS_2A Prisoner’s 2A Wild The Cultured Abalone
CI_SCI_PRIS_1B Prisoner’s 1B Wild The Cultured Abalone
CI_SCI_PRIS_2B Prisoner’s 2B Wild The Cultured Abalone
RM_0M_SW Rancho Marina 0M SW Wild The Abalone Farm
RM_0M_SW_#1 Rancho Marina 0M SW #1 Wild The Abalone Farm
PSN_0M_#2 Pt. Sierra Nevada 0M #2 Wild The Abalone Farm
PSN_0M_#1 Pt. Sierra Nevada 0M #1 Wild The Abalone Farm
CARMEL_0M_2 Carmel 0M 2 Wild American Abalone
AMER_DRAIN_1A American Abalone Drain 1A Farm Carmel
AMER_DRAIN_2A American Abalone Drain 2A Farm Carmel
AMER_DRAIN_1B American Abalone Drain 1B Farm Carmel
AMER_DRAIN_2B American Abalone Drain 2B Farm Carmel
AMER_0M_OUT_1A American Abalone 0M Outfall 1A Farm Carmel
AMER_0M_OUT_2A American Abalone 0M Outfall 2A Farm Carmel
AMER_0M_OUT_1B American Abalone 0M Outfall 1B Farm Carmel
AMER_0M_OUT_2B American Abalone 0M Outfall 2B Farm Carmel
TAF_DRAIN_DUP2B The Abalone Farm Drain Dup 2B Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_2C The Abalone Farm Drain Dup 2C Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_DUP2D The Abalone Farm Drain Dup 2D Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_1D The Abalone Farm Drain Dup 1D Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_DUP2A The Abalone Farm Drain Dup 2A Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_DUP1A The Abalone Farm Drain Dup 1A Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_1B The Abalone Farm Drain Dup 1B Farm Pt. Sierra Nevada/Rancho Marina
TAF_DRAIN_1C The Abalone Farm Drain Dup 1C Farm Pt. Sierra Nevada/Rancho Marina
CAB_N.OUT_1A The Cultured Abalone North Outfall 1A Farm Santa Cruz Islands
TCA_N.OUT_1B The Cultured Abalone North Outfall 1B Farm Santa Cruz Islands
CAB_N.OUT_1C The Cultured Abalone North Outfall 1C Farm Santa Cruz Islands
CAB_N.OUT_1D The Cultured Abalone North Outfall 1D Farm Santa Cruz Islands
TCA_S.OUT_1A The Cultured Abalone South Outfall 1A Farm Santa Cruz Islands
TCA_S.OUT_1B The Cultured Abalone South Outfall 1B Farm Santa Cruz Islands
CAB_S.OUT_1C The Cultured Abalone South Outfall 1C Farm Santa Cruz Islands
CAB_S.OUT_1D The Cultured Abalone South Outfall 1D Farm Santa Cruz Islands

Results:
qPCR Report (PDF): Sam_2015-12-28 15-44-35_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-28 15-44-35_CC009827.pcrd

Overall, data looks good. Will enter copy numbers into the Ab Endo master sheet for later analysis (Google Sheet): Ab Endo Samples

 

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qPCR – RLOv DNA Helicase Standard Curve Check Repeat

Yesterday’s check of the RLOv DNA helicase standard curve using a freshly made 3 copy sample didn’t look perfect, so I’m re-running to see if things will tighten up a bit.

Repeated yesterday’s run with no changes.

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151223 – qPCR RLOv DNA Helicase Curve Check

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

Results:
qPCR Report (PDF): Sam_2015-12-24 11-02-59_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-24 11-02-59_CC009827.pcrd

 

Overall, things are looking better than yesterday’s run: better reps, better efficiency and better R^2. Will move forward with beginning to validate this qPCR assay, as well as use for some other sample analysis that Carolyn has in mind (comparing RLOv vs. WS levels in abalone collected from wild sites in California).

 

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qPCR – RLOv DNA Helicase Standard Curve Check

Since the standard curve for this assay was a bit wonky at the low copy number end the last time I ran it, I made a fresh 1:10 dilution of the 3 copies component of the curve (100μL of 30 copy sample in 900μL TE).

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20151223 – qPCR RLOv DNA Helicase Curve Check

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

Results:
qPCR Report (PDF): Sam_2015-12-23 13-57-01_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-23 13-57-01_CC009827.pcrd

 

The efficiency & R^2 values look pretty solid, but the spacing between the 300 copy (Cq ~ 32 in the amplification plot) and the 30 copy (Cq ~ 34 in the amplification plot) samples is a bit too tight for my liking. Additionally, the reps for the 3 copy sample are very poor.

Will repeat to see if I can tighten things up…

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qPCR – Black Abalone DNA with Varying Levels of RLO/RLOv

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.

Earlier today I identified samples at three different histology scoring levels of RLO: 0, 1, & 2.

Here’s the list of samples that will be qPCR’d. There were only eight samples that had histology scores of 2 in both PE and Dg.

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
06:5-18 06:5-35 06:5-31
06:5-30 06:6-32 06:5-32B
06:50-04 06:6-39 06:6-46
06:50-05 06:6-42 06:6-49
07:12-01 06:6-44 08:3-05
07:12-02 06:6-52 08:3-07
07:12-03 06:6-54 08:3-15
07:12-04 06:50-08 08:3-16
07:12-07 06:50-10
07:12-09 07:12-18

 

Ran qPCR using the RLOv DNA helicase standard curve from 20151106.

All samples were run in duplicate on the CFX96 (BioRad).

Master mix calcs are here: 20151120 – qPCR RLOv Black Abs

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

Results:
qPCR Report (PDF): Sam_2015-11-20 15-00-27_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-11-20 15-00-27_CC009827.pcrd

Quick summary of the results:

  • 50% of the RLO/RLOv 0 score samples are positive for RLOv DNA helicase. Will talk to Carolyn to see if she has withering syndrome qPCR data for these samples to compare RLOv-positive samples with WSN-positive samples. If not, will run withering syndrome qPCR.
  • All RLO/RLOv 1 & 2 scored samples are positive for RLOv DNA helicase
  • All RLO/RLOv 2 scored samples come up before the standar curve; these should be diluted and re-run.
  • Standard curve isn’t perfect (the 3 copy sample is throwing it off).

 

STANDARD CURVE AMP & SCATTER PLOTS

 

 

RLO/RLOv 0 AMP PLOTS

 

 

RLO/RLOv 1 AMP PLOTS

 

 

RLO/RLOv 2 AMP PLOTS

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Data Management – Black Abalone Histology Scores

As part of the qPCR validation for the withering syndrome phage (RLOv) project, I needed to identify (and, eventually locate) samples that are infected with varying levels of RLOv. This is probably the most time consuming aspect of the project.

I found the histology scoring sheets and added them to an existing Google Sheet that Lisa had partially completed a few years ago: Black Abalone: Expt 1 – WS & Phage

To save time, I only entered the scores into the spreadsheet and did not enter any extra info (like Sex or Coccidia).

Having this data in a single, digital format will allow me to sort the data, to quickly & easily select the appropriate samples with varying levels of RLOv (categorized as “New” on the sheet).

Here are links to pics of the histology scoring sheets for reference:

Next up will be to actually track down the physical samples. This will be a bit of a daunting task…

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PCR – RLOv In-situ Hybridization (ISH) Probes

Ran probe-labeling PCRs to use in in-situ hybridization (ISH) using the PCR DIG Probe Sysnthesis Kit (Roche). Generated PCR probes for using the following BamHI-linearized plasmids:

  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_tail_fiber

The Roche protocol recommends using only 10pg of plasmid DNA for probe labelling. As such, all three probes were diluted 1:10,000. A 1:1000 (999μL H2O + 1μL of plasmid) was made first. Then a 1:10 dilution was made (90μL H2O + 10μL from 1:1000 dilution of plasmid).

Additionally, I ran half reactions to conserve kit components. Roche recommends 50μL reactions; I ran 25μL and scaled all components appropriately.

All reactions were set up on ice and run in 0.2mL strip-cap PCR tubes.

Reaction calculations are here (Google Sheet): 20151109 – RLOv ISH Probe PCRs

Cycling params:

  1. 95C – 5mins
  2. 95C – 15s
  3. 55C – 15s
  4. 72C – 30s
  5. Go to Step 2, repeat 39 times.
  6. 72C – 10mins

After the PCR, 5μL of each reaction was run on a gel.

Results:

Hyperladder I (Bioline)

PCR DIG probe labelling products run on 1.1% agarose 1x TBE gel stained w/EtBr. A ‘+’ indicates DIG reaction, while a ‘-‘ indicates no DIG in reaction.

Two reactions were run for each plasmid: one with the DIG label (indicated by a ‘+’) and one without (indicated by a ‘-‘). If the labeling was successful, the PCR products from those reactions containing DIG will be larger (i.e. migrate slower) than those without. That is exactly what we see in each of the three potential ISH targets.

So, we now have three ISH probes ready for action! Will proceed with making fresh ISH buffers and ISH.

Probes were transferred to 0.5mL snap cap tubes and stored in my -20C box.

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Restriction Digestions – pCR2.1/RLOv Plasmids

Set up restriction digestions to linearize the pCR2.1/RLOv plasmids in preparation for ISH probes and qPCR standard curves. Used BamHI (NEB), since it doesn’t cut in any of the RLOv sequences and cuts one time in pCR2.1-TOPO (Invitrogen).

PLASMID Vol for 1.5μg (μL) H2O to 40μL
pCR2.1/RLOv_DNA_helicase 21.4 18.6
pCR2.1/RLOv_head_to_tail 11.1 28.9
pCR2.1/RLOv_membrane_gene_1 12.2 27.8
pCR2.1/RLOv_membrane_gene_2 14.3 25.7
pCR2.1/RLOv_tail_fiber 20 20

 

Digestion Master Mix

REAGENT SINGLE REACTION (μL) x 5.5 (μL)
Plasmid 40 NA
10x Buffer 3.1 (NEB) 5 27.5
BamHI (NEB) 1 5.5
H2O 4 22
TOTAL 50 Add 10μL to each tube

Digests were incubated at 37C for 1hr in PTC-200 thermal cycler (MJ Research); no heated lid.

Ran 3μL of undigested plasmid and 10μL of linearized plasmid on 0.8% agarose 1x TBE gel stained w/EtBR.

Results:

Hyperladder I (Bioline)

U = Undigested; Bam = BamHI digest

Besides the funky way this gel ran, the digests look to be complete.

Will quantify remaining linearized plasmids with a dye-based method for accurate quantification and then proceed with the making ISH probes (membrane genes and tail fiber gene) or qPCR standard curves (DNA helicase and head-to-tail).

 

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Sanger Sequencing Analysis – pCR2.1/RLOv Clones

Sequencing results from the samples that were submitted to Genewiz on Friday have come back:

  • SW01    RLOv_DNA_Helicase-M13F_-21_
  • SW02    RLOv_head_to_tail-M13F_-21_
  • SW03    RLOv_membrane_gene_1-M13F_-21_
  • SW04    RLOv_membrane_gene_2-M13F_-21_
  • SW05    RLOv_tail_fiber-M13F_-21_
  • SW06    RLOv_DNA_Helicase-M13R
  • SW07    RLOv_head_to_tail-M13R
  • SW08    RLOv_membrane_gene_1-M13R
  • SW09    RLOv_membrane_gene_2-M13R
  • SW10    RLOv_tail_fiber-M13R

The data (10-313205054_ab1.zip) has been stored in the following location: backupordie/sequencing_data/Sanger.

Sequences were loaded into Geneious (v.9.0.2). Vector sequences were trimmed/annotated using the Trim Ends with UniVec feature in Geneious.

Each clone was sequenced once from each direction, so the two sequences generated from each clone were mapped to the original sequence from which the primers were designed using Geneious Mapper.

The Geneious analysis was exported and saved in the following location:

backupordie/Sam/Sequencing_Analysis/Sanger/20151026_RLOv_clones_Sanger_analysis.geneious

Results:

Each clone’s sequence matches that of the source sequence, so we’re good to go!

Will proceed with dye-based quantification of each plasmid. Will then proceed with developing ISH probes (membrane genes 1 & 2, tail fiber gene) or qPCR standard curves (DNA helicase, head-to-tail).

In the alignments below, the reference sequence is highlighted in light yellow. The two electropherograms are align below the reference. The grey line in the consensus sequence indicates any sequence disagreements by placement of a black mark at the position. However, the sequences all match, so there are no black marks in the regions between the identified vector sequences (red annotations below each electropherogram).

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