Tag Archives: RLOv

Data Aggregation – WS RLO and RLOv DNA Helicase qPCR and WS RLO Infection Intensities

Carolyn asked me to send her the data described above.

RLOv DNA helicase qPCR data were grabbed from the qPCR I ran on 20160106.

The qPCR data for withering syndrome RLO were culled from these three different spreadsheets:

 

The summary is below. I have emailed a copy of the spreadsheet to Carolyn.

Google Sheet: 20160404_Summary_RLO_RLOvDNAhelicase_qPCR_HistoIntensities

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qPCR – XenoCal Prophage Portal Primers on RLO/RLOv positive/negative samples

Stan Langevin and Carolyn wanted to see if this particular gene was found in the withering syndrome (RLO) or phage (RLOv) genomes. I previously identified 10 samples of each of the following combinations:

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

Master mix calcs are here (Google Sheet): 20160331 – qPCR Black Ab 08:13 XenoCal phage portal check

All samples were run in duplicate.

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

Results:
qPCR Report (PDF): Sam_2016-03-31 08-51-11_CC009827.pdf
qPCR Data File (CFX): Sam_2016-03-31 08-51-11_CC009827.pcrd

The results are definitely interesting!

Quick summary: Amplification only seen in RLO+/RLOv- samples!

Oddly, there is no amplification in the other group of RLO+ samples (RLO+/RLOv+). Based on the fact that there is amplification in RLO+/RLOv- samples, which implies this prophage portal gene is present in withering syndrome, we would expect to also have amplification in the other group of samples that are positive for withering syndrome.

Compiled qPCR data with WSN1, RLOv_DNA_helicase, and XenoCal prophage portal gene (Google Sheet): 20160331_qPCR_summary_RLO_RLOv_pos_negs

 

Amplification Plots (PINK= RLO+/RLOv-, BLUE = RLO-/RLOv-; GREEN = RLO-/RLOv+; BLACK = RLO+/RLOv+)

 

Melt Curve Plots (see amplification plots for color scheme)

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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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Data Analysis – RLOv DNA Helicase qPCR Limit of Detection Calcs

I previously ran three qPCR plates (20160121, 20160122, & 20160125) with 20 reps of each of the following copy numbers to determine the limit of detection (or, analytical sensitivity) of this qPCR assay: 30, 10, 3, 1

Additionally, I determined the average baseline threshold to set (580.5), based on eight qPCRs (see 20160128).

Performed calculations and determined limit of detection (defined as >95% of samples produced amplification) for this assay is three copies. The single copy sample amplified 88% of the time. This also means that our cycle threshold cut-off for this qPCR assay should be 37.9, as the mean Cq for three copies was 37.33, with a standard deviation of 0.53.

The data and calculations can be seen below (Google Sheet – scroll to the right to see calcs):

Google Sheet: RLOv_DNA_helicase_LoD_calcs

 

 

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Data Analysis – qPCR Baseline Threshold Determination for RLOv DNA Helicase qPCR Assay

Idetermined a qPCR baseline threshold to use for the withering syndrome RLOv DNA helicase qPCR assay in the following fashion.

I adjusted baseline threshold values of the following eight qPCR runs so that the mean Cq  values for each component of the standard curves (per plate) across plates were within 0.5 Cqs and averaged the baseline thresholds.

The average baseline threshold was 580.5.

This value will be manually applied to all past and future RLOv DNA helicase qPCR runs that were conducted on the Friedman Lab CFX96.

FILE ADJUSTED THRESHOLD
Sam_2016-01-25 10-48-06_CC009827.pcrd 609
Sam_2016-01-22 10-15-55_CC009827.pcrd 569
Sam_2016-01-21 15-12-31_CC009827.pcrd 576
Sam_2016-01-06 16-01-36_CC009827.pcrd 550
Sam_2015-12-28 15-44-35_CC009827.pcrd 613
Sam_2015-12-28 11-20-16_CC009827.pcrd 614
Sam_2015-12-24 11-02-59_CC009827.pcrd 531
Sam_2015-12-23 13-57-01_CC009827.pcrd 582

 

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Continuing RLOv DNA Helicase qPCR assay validation.

This is the third of three plates to establish the assay’s limit of detection.

The first plate was run 20160121.

The second plate was run 20160122.

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.
  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

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

Results:
qPCR Report (PDF): Sam_2016-01-25 10-48-06_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-25 10-48-06_CC009827.pcrd

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Continuing RLOv DNA Helicase qPCR assay validation.

This is the second of three plates to establish the assay’s limit of detection. The first plate was run yesterday (20160121).

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.

  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

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

Results:
qPCR Report (PDF): Sam_2016-01-22 10-15-55_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-22 10-15-55_CC009827.pcrd

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qPCR – RLOv DNA Helicase Assay Limit of Detection

Beginning RLOv DNA Helicase qPCR assay validation.

This is the first of three plates to establish the assay’s limit of detection.

The limit of detection assessment is conducted in the following fashion:

  • Three plates of qPCRs; each plate run on different days.

  • On each plate; 20 reps each of the following standard curve copy numbers: 30, 10, 3, 1

Master mix calcs (Google Sheet): 201600121 – qPCR RLOv DNA Helicase Promega LoD-1

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

Results:
qPCR Report (PDF): Sam_2016-01-21 15-12-31_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-21 15-12-31_CC009827.pcrd

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

Repeated the qPCR from 20151120 because the last attempt I did not have samples that had been shown to be clean of RLO via qPCR; had used histology scoring to identify RLO-negative samples. Additionally, many of the samples that produced amplification were out of range of the standard curve (most came up too early) and require dilution to be properly assessed.

RLO/RLOv 0 RLO/RLOv 1 RLO/RLOv 2
08:4-3 06:5-35 06:5-31
08:4-4 06:6-32 06:5-32B
08:4-5 06:6-39 06:6-46
08:4-6 06:6-42 06:6-49
08:4-7 06:6-44 08:3-05
08:4-8 06:6-52 08:3-07
08:4-9 06:6-54 08:3-15
08:4-10 06:50-08 08:3-16
08:4-11 06:50-10
08:4-12 07:12-18

Made 1:1000 dilutions of all of the RLOv 1 and RLOv 2 samples.

Master mix calcs are here (Google Sheet): 20160106 – qPCR RLOv black abs

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

All samples were run in duplicate.

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

Results:
qPCR Report (PDF): Sam_2016-01-06 16-01-36_CC009827.pdf
qPCR Data File (CFX96): Sam_2016-01-06 16-01-36_CC009827.pcrd

Summary:

Standard curve looks solid.

RLO-negative samples are all negative for RLOv DNA helicase.

1:1000 dilutions of RLOv 1 and RLOv 2 all amplified within the range of the standard curve.

 

RLO Negative Amplification Plots

 

RLO 1 Amplification Plots

 

RLO 2 Amplification Plots

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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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