Category Archives: 2011 Samples

2011 Samples

qPCR – RLOv DNA helicase and XenoCal prophage on Ab Endo Water Filters

Stan Langevin was interested in seeing if the RLOv (phage) and/or the prophage portal genes were detectable in water samples from Lisa’s Ab Endo project.

Ran qPCR on the following samples that Lisa selected:

DNA from water filters collected in 2010. DNA isolated 20120111:

  • CP 0M A
  • CP 0M B
  • MA 0M A
  • MA 0M B
  • PSN 0M A
  • PSN 0M B
  • RM A
  • RM B

DNA from water filters collected in 2011. DNA isolated 20140822:

  • AM Drain 2B
  • PCI SRI PC 1B

RLOv_DNA_helicase master mix calcs are here (Google Sheet): 20161213 – qPCR RLOv DNA Helicase

XenoCal prophage master mix calcs are here (Google Sheet): 20161213 – qPCR XenoCal phage portal

RLOv_DNA_helicase standard curve from 20151224.

All samples were run in duplicate. Plate layout, cycling params, etc. can be seen in the qPCR Report below.

Results:

RLOv_DNA_helicase
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_RLOv_helicase.pcrd

 

XenoCal prophage
qPCR Report (PDF): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pdf
qPCR Data File (CFX): Sam_2016-12-13 14-52-05_CC009827_XCprophage.pcrd

 

  • RLOv DNA helicase amplified in all samples EXCEPT the two samples from 2011. These two samples were negative for the RLO (see Ab Endo sheet “water 2011″).
  •  XC prophage amplfied inconsistently (i.e. replicates did not match/amplify) in only three samples. Additionally, the melt curve of one of those samples differs from the other two. Based on the inconsistencies in technical reps, I should probably repeat this, but technical reps across all of the RLOv DNA helicase samples are very tight, suggesting that my technique was fine (it would be odd if my technique faltered only on ALL of the XC prophage samples)…

 

RLOv DNA HELICASE

 


 

XENOCAL PROPHAGE

 

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Sample ID- XenoCal Prophage Portal Tests

Now that the XenoCal prophage portal primers appear to be in working order, Carolyn wants me to test them out on 10 samples with the following status':

  • RLO-/RLOv-
  • RLO-/RLOv+
  • RLO+/RLOv-
  • RLO+/RLOv+

In order to quickly identify samples with these qualifications, I ran a SQL query on the following spreadsheet that contain qPCR data for both withering syndrome (RLO) and the phage (RLOv):

I saved the following worksheets from the above Google Sheet as CSV files:

  • water 2010
  • water 2011

These were imported to SQLite as I’ve previously done.

The two sheets were renamed for use in SQLite, respectively:

  • AbEndoWater2010
  • AbEndoWater2011

Here are the four queries I ran to obtain the four combinations of RLO/RLOv samples listed above

RLO-/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq"=0

 

RLO-/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq"=0 AND "RLOv_mean_Cq">0

 

RLO+/RLOv-

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq"=0

 

RLO+/RLOv+

sqlite> SELECT '2011_H2O', "DNA Tube Label", "Mean Cq", "RLOv_mean_Cq" FROM AbEndoWater2011 WHERE "Mean Cq">0 AND "RLOv_mean_Cq">0

 

Results:

It looks like we do not currently have 10 samples that are RLO+/RLOv-. I will contact Carolyn to see if she happens to know of any samples that are RLO+, but do not contain (or, should not) any RLOv.

The full list of results can be seen in the Google Sheet below.

Google Sheet: 20160322_RLO_RLOv_pos_negs

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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 – 2011 Ab Endo Water Filters

Ran qPCRs on remaining 2011 water filter DNA samples. All samples were from The Cultured Abalone farm, but some samples are abbreviated “CAB”, while others “TCA”. Samples were extracted 20131001 and 20131120.

Master mix calcs: 20150702 – qPCR WS Ab Endo H2O Filters

All samples were run in duplicate. See qPCR Report in Results below for cycling params.

Standard curve was p18RK7 from 20120731.

Baseline threshold was set to 580, as determined by Lisa.

Results:
qPCR Report (PDF): Sam_2015-07-02 09-45-05_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-07-02 09-45-05_CC009827.pcrd

Data entered in the Ab Endo Samples Google Sheet.

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qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20150622 – qPCR WS Ab Endo H2O Filters

All samples were run in duplicate. See the qPCR Report (in Results below) for plate layout, cycling params, etc.

Standard curve was p18RK7 from 20120731.

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2015-06-22 14-25-45_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-06-22 14-25-45_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet.

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qPCR – Withering Syndrome ddPCR Comparison Repeat

Repeated the qPCR from yesterday due to inconsistent results when compared to the previous qPCR data (see table in yesterday’s notebook entry), but used the stock DNA for template.

These samples were initially quantified as part of the Ab Endo Project (Ab Endo 2011 Water Filter DNA samples). Here’s the list of samples:

  1. CI SRI CP 1A
  2. CARMEL +500M 2
  3. CI SRI CP 2B
  4. CI SRI CP 2A
  5. CI SRI CP 1B
  6. CARMEL +500M 1

Master mix calcs are here: 20150415 – qPCR WS Ab Endo vs ddPCR

Standard curve was p18RK7 (from 20120730).

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

Results:
qPCR Report (PDF): Sam_2015-04-16 12-14-37_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-04-16 12-14-37_CC009827.pcrd

TABLE – Summary of qPCRs and ddPCR data

Sample Number Sample Name Initial qPCR Copies ddPCR Copies Yesterday’s qPCR Copies Today’s qPCR Copies
AB01 CI SRI CP 1A 0 3.8 0 0
AB02 CARMEL +500M 2 1.57 8.7 1.3 1.05
AB03 CI SRI CP 2B 147 104 59.2 201
AB04 CI SRI CP 2A 0 175 83.1 0.68
AB05 CI SRI CP 1B 74.8 2.8 0 101
AB06 CARMEL +500M 1 1.08 4.9 0 0.37

Today’s qPCR data matches up very well with the initial qPCR data.

Clearly, there is something odd about the aliquots that were sent to Alice (and back) for use in ddPCR. I can’t really fathom what happened. Will contact Alice and see if she’d like to try out new aliquots of these samples to see how the ddPCR turns out (again).

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qPCR – Withering Syndrome ddPCR Comparison

Performed qPCR on the same exact samples previously sent to Alice Nguyen at the Marine Science Institute for digital droplet PCR (ddPCR) to compare our results to hers. These samples were previously quantified as part of the Ab Endo Project (Ab Endo 2011 Water Filter DNA samples). Here’s the list of samples that were sent to (and back from) Alice:

  1. CI SRI CP 1A
  2. CARMEL +500M 2
  3. CI SRI CP 2B
  4. CI SRI CP 2A
  5. CI SRI CP 1B
  6. CARMEL +500M 1

Master mix calcs are here: 20150415 – qPCR WS Ab Endo vs ddPCR

Standard curve was p16RK7 (from 20120730).

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

Results:
qPCR Report (PDF): Sam_2015-04-15 15-38-13_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-04-15 15-38-13_CC009827.pcrd

Table – Summary of qPCRs and ddPCR data

Sample Number Sample Name Initial qPCR Copies ddPCR Copies Today qPCR Copies
AB01 CI SRI CP 1A 0  3.8  0
AB02 CARMEL +500M 2 1.57  8.7  1.3
AB03 CI SRI CP 2B 147  104  59.2
AB04 CI SRI CP 2A 0  175  83.1
AB05 CI SRI CP 1B 74.8  2.8  0
AB06 CARMEL +500M 1 1.08  4.9  0

The table certainly shows some inconsistency, both between ddPCR and qPCR. Additionally, there is also inconsistency between the initial qPCRs and today’s qPCRs. Due to the limited sample volumes remaining for these sub-samples that were sent to Alice and then returned to us, I will repeat the qPCR using the stock DNA and see how that compares.

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qPCR – 2011 Ab Endo Water Filters

Master mix calcs are here: 20141104 – qPCR 2011 Ab Endo Water Filters

All samples were run in duplicate. See the qPCR Report (in Results below) for plate layout, cycling params, etc.

Standard curve was p18RK7 from 20120731.

Baseline threshold set to 580, based on calculations by Lisa.

Results:

qPCR Report (PDF): Sam_2014-11-04 14-25-07_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-11-04 14-25-07_CC009827.pcrd

All data was entered in to the Ab Endo Samples spreadsheet. Samples that require repeating were highlighted in orange.

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