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

 

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

qPCR – RLOv DNA Helicase 2010 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 the nearest wild abalone site.

DNA samples used were extractions from water filters collected for the Ab Endo Project in 2010.

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 2010 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
painted-SCI_B Painted Cave SCI B Wild The Cultured Abalone
painted-SCI_A Painted Cave SCI A Wild The Cultured Abalone
prison-SCI_B Prisoner’s SCI B Wild The Cultured Abalone
prison-SCI_A Prisoner’s SCI A Wild The Cultured Abalone
TCA_out_East_A The Cultured Abalone Outfall East A Farm Santa Cruz Islands
TCA_out_East_B The Cultured Abalone Outfall East B Farm Santa Cruz Islands
TCA_out_West_A The Cultured Abalone Outfall West A Farm Santa Cruz Islands
TCA_out_West_B The Cultured Abalone Outfall West B Farm Santa Cruz Islands
TAF_ND_A1 The Abalone Farm North Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A1 The Abalone Farm South Drain A1 Farm Pt. Sierra Nevada/Rancho Marina
TAF_SD_A2 The Abalone Farm South Drain A2 Farm Pt. Sierra Nevada/Rancho Marina
RM_A Rancho Marina A Wild The Abalone Farm
RM_B Rancho Marina B Wild The Abalone Farm
AmA_Drain_A1 American Abalone Drain A1 Farm Carmel
AmA_Drain_A2 American Abalone Drain A2 Farm Carmel
AmA_Drain_B1 American Abalone Drain B1 Farm Carmel
AmA_Drain_B2 American Abalone Drain B2 Farm Carmel
PSN_0M_A Pt. Sierra Nevada 0M A Wild The Abalone Farm
PSN_0M_B Pt. Sierra Nevada 0M B Wild The Abalone Farm
CP_0M_A1 Carmel 0M A1 Wild American Abalone
CP_0M_B Carmel 0M B Wild American Abalone
CP_0M_A Carmel 0M A Wild American Abalone

Results:
qPCR Report (PDF): Sam_2015-12-28 11-20-16_CC009827.pdf
qPCR Data File (CFX96): Sam_2015-12-28 11-20-16_CC009827.pcrd

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

qPCR – p16RK7 Curve Old Primers and Probe Test, Lisa Sample Check

Ran qPCRs testing the “old” primers/probe (IDT primers, Biosearch Technologies Probe) using ABI 2x qPCR master mix and Promega 2x qPCR master mix.

The curve used was the p16RK7 from 20120731. This was used because it worked perfectly on 20140418.

Also ran a qPCR with all ABI components along with the following set of samples that Lisa needed checked for positive/negative of withering syndrome:

2010 Water Filter DNAs (Site 8 extracted 20120228 and Site 3 extracted 20120229 by me):

  • SNI Site 8 0M Rep. 1
  • SNI Site 8 +100M Rep. 1
  • SNI Site 8 -100M #1
  • SNI Site 8 +500M #1
  • SNI Site 8 -500M #1
  • SNI Site 3 0M #1
  • SNI Site 3 +100M #1
  • SNI Site 3 -100M #1
  • SNI Site 3 +500M #1
  • SNI Site 3 -500M #1

Master mix calcs are here: 20140422 – qPCR p16RK7 Curve Old Probe

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

All samples were run in duplicate.

Results:

qPCR Report (PDF): Sam_2014-04-22 15-50-08_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-04-22 15-50-08_CC009827.pcrd

NOTE: Since this used a different polymerase (ABI) than what has been used in the past for this assay (Bioline), I did NOT adjust the baseline threshold. A new baseline threshold will have to be decided upon.

The qPCR run with the new probe/primer set worked wonderfully! The qPCRs with the old primer/probes failed miserably!

Here are the amplification curves and standard curve graph from the new primer/probe sets using the ABI master mix. The brown colored curves are the standard curve. The blue colored curves are some of Lisa’s samples.

Below are the other TWO curves (yep, both of them!) run with the old primer/probe set. Purple colored curves are Promega master mix, green colored curves are ABI master mix.

qPCR – p16RK7 Curve With SPUD, Repeat Lisa 2010 Water Filter DNA Subset

Ran qPCR with the p16RK7 curve using the new ABI reagents to test SPUD performance now that we have a fully functional withering syndrome qPCR assay again.

Also repeated the following 2010 Water Filter DNAs that were run yesterday, due to lack of amplification in one of the duplicates (likely due to very late amplification):

  • SNI Site 8 +100M #1
  • SNI Site 3 -100M #1
  • SNI Site 3 -500M #1

Master mix calcs are here: 20140423 – qPCR p16RK7 Curve WSN1 SPUD

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

Results:

qPCR Report (PDF): Sam_2014-04-23 13-07-58_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-04-23 13-07-58_CC009827.pcrd

For Lisa’s samples, only the SNI Site 3 -100M #1 showed any amplification (mean Cq of 39.31).

The SPUD assay still shows inhibition at higher concentrations of the standard curve (>= 3e4 copies). Possible that the SPUD probe is dying (the working stock has been through many freeze/thaw cycles which could definitely be affecting its performance…). This is slightly disconcerting, but I’m just happy that the standard curve is functional once again!!

qPCR – Withering Syndrome qPCR Assay Sample Checks

Ran qPCR on water filter samples isolated on 20120127 in an attempt to find water samples that have a higher copy number than Nate’s existing samples for use in the withering syndrome qPCR assay validation.

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

Standard curve was the p16RK7 NcoI-linearized curve made on 20120730.

Baseline threshold was set to 400 and cycles to analyze was set to 41.

Results:

qPCR Data File (CFX96):Sam_2012-10-17 15-49-02_CC009827.pcrd

qPCR Report (PDF):Sam_2012-10-17 15-49-02_CC009827.pdf

Everything looked good. Will use sample TAF SD A2 as the high copy number water sample for the withering syndrome qPCR assay.

DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.

DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.

DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.

DNA Extraction – Abalone Water Filters from Summer 2010

Extracted DNA from water filters collected during Summer 2010 from various locations using the DNeasy Kit. Based on extraction methodology tests (see 20111221), filter were each cut into ~13 strips and placed in a 1.5mL snap cap tube containing 400uL of Buffer AL and 50uL of Proteinase K. The volumes of both reagents are double the kit recommendation. Samples were vortexed thoroughly and incubated at 56C O/N. After incubation, 400uL (twice the volume in the Qiagen protocol) of 100% EtOH was added to each tube and vortexed thoroughly. The kit protocol was followed for the remainder of the procedure. Samples were eluted with 100uL of Buffer AE and stored @ 4C. Spreadsheet indicating which samples were extracted is here.