My Notebook is now here: http://onsnetwork.org/sjwfriedmanlab















20101215

qPCR - Vp Assay w/SPUD IPC Kit

This is repeating Robyn's qPCR tests with the SPUD IPC assay to see if I get the same, weird results (IPC detection coming up in all IPC-negative samples at a late Ct, while in IPC-positive samples it comes up at a very early Ct) as her.

Fresh working stocks of the SPUD IPC Kit were made and used in the qPCR master mix.

Vp dilution series DNA was made by Robyn and extracted 20101119.

Oyster DNA was made by Robyn and extracted 20101119.

Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR report (see Results).


Results:
qPCR Report (PDF).

I get the same results as Robyn. This suggests that the SPUD IPC is not viable with this assay. It also suggests that the SPUD IPC is not good entirely, however, to my knowledge, the SPUD IPC has not been tested in any other type of qPCR assay to see if this problem is assay specific or a problem with the SPUD IPC.





20101208

qPCR - Black Abalone Dg Experiment 1, Rep 1 with New Normalizing Gene, NEC1_RAT

Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR report (see Results).

Results:



20101206

qPCR - Abalone Virus Primer Checks

Tested the 8 abalone virus primers that were generated from the abalone SOLiD data whereby the Carmel Control samples were subtracted from the Carmel Exposed samples. Here's the sheet with the primers used (highlighted in yellow) and their corresponding short names.

Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR report (see Results).

cDNA made on 20080612 from abalone Dg samples 08:3-6 (term) and 07:12-17 (mid) was used. Samples were selected by Lisa based on high score values for the presence of the "New" bug observed. 07:12-17 had a "New" score of 3 and 08:3-6 had a "New" score of 2.5. Scores and other data can be found here (data is not public; permission required to view/access; contact me if you need to view and are unable to).

Results:
qPCR Report (PDF).

Two primer sets did NOT amplify: AbV02 (EU296611, hemagglutinin)and AbV08 (NC_008296, tRNA-Ala).
Two primer sets showed good amplification and perfect melt curves: AbV03 (GU934326, neomycin phosphotransferase) and AbV06 (M38308, RNA Polymerase).
The remaining primer sets should be subjected to optimization (anneal temp. gradient and Mg2+ gradient).



20101202

qPCR - Hemat Std. Curves Validation #1 and #2


Performed qPCR on 2 of 3 standard curves from 20101007. Each dilution in each curve was subjected to 4 replicates. The third curve will be tested by Lisa, per the requirements for this validation. Master mix calcs are here. Plate layout, cylcing params, etc. can be found in the qPCR Report (see Results).


Results:
qPCR Report (PDF).

Both curves look pretty good. Combined, they exhibit a R2 = 0.990.

Curve set #1 exhibits a R2 = 0.998. The sample-to-sample accuracy does drift a bit at the lowest three copy numbers (10, 3, 1).

Curve set #2 exhibits a R2 = 0.988. The sample-to-sample accuracy does drift a bit at the lowest three copy numbers (10, 3, 1), but moreso than the curve set #1 and is likely what leads to the lower R2 value.

Will have to wait until Lisa runs the curve set #3 to see how these three curves compare and if they are satisfactory for the Hematadidium validation assay.


20101201

qPCR - Hemat NcoI Probe Comparison

Due to the poor appearance of the curves run yesterday on the freshly prepared plasmids, have decided to try one of the new curves with all three of the Hemat probes that we have in hopes of getting better (e.g. useable!) results. Master mix calcs are here. Plate set up, cycling params, etc can be seen in the qPCR Report (see Results).


Results:

qPCR Report (PDF) is here.

Well, it turns out the problem WAS the probe! Clearly, when I did this comparison before, I accidentally selected the incorrect probe as being the best. So, the probe breakdown is like this:

Hemat_p: Good
Hemat_p191/214: BEST
Hemat_p214: BAD!!

In the qPCR Report, the Hemat_p214 probe samples are those that are the samples exhibiting extremely low fluorescence levels (rows E & F).




20101130

qPCR - Hemat NcoI New Standard Curves from yesterday

Master mix calcs are here. Plate set up, cycling params, etc can be seen in the qPCR Report (see Results).

Results:
qPCR Report (PDF) is here.

The curves look poor, just like the others I've diluted. This is odd because I've made dilutions of curves using different solvents (water, TE) and using different stocks to make the curve dilutions. Is it possible that the probe that worked the best with the old, previously made curves might not work with these newer curves that I've made? It doesn't seem logical, but I can rule out poor technique being issue because I've made curves with the other projects I've worked on (Vt, Vp) without issue. I will test out the other two probes with these new curves that I've made and see what effect, if any, the different probes have on my results...






20101129

New Std. Curve Samples

Used purified, linearized Hematadidium plasmid DNA from 20101109. Here are the dilution calcs for each curve:

Hemat 30
Hemat 40

Dilutions were done with TE.


20101109

Restriction Digestions - Hemat plasmids isolated 20101022

Performed RE digests on the 30 and 40 Hemat plasmids (1ug) using NcoI. Digests were incubated 1hr @ 37C. Samples were heat inactivated @ 65C for 20 mins. Samples were allowed to cool and then treated with antartic phosphatase according to NEB protocol to prevent religation of the vector. Samples were then cleaned up using Qiagen's MiniElute PCR Purification Kit, eluted with 10uL of EB and spec'd.

Results:

external image 20101109%20Hemat%20NcoI%20phosph%20purified.JPG


Each sample was spec'd 4 times in order to get an accurate concentration. Concentrations look OK (~320ng total recovery) and the OD260/280 look very good.

Hemat 30 = 34.085 ng/uL
Hemat 40 = 31.115 ng/uL
Will test these for use as standard curves.




20101102

Restriction Digestions - Hemat plasmids isolated 20101022

Performed RE digests on the 30 and 40 Hemat plasmids (1ug) using NcoI. Digests were incubated 1hr @ 37C.


Results:

Gel was entirely blank. Realized I forgot to add the DNA. Duh. Rookie move. Will repeat.







20101028

Restriction Digestions - Hemat plasmids isolated 20101022

Performed RE digests on the 30 and 40 Hemat plasmids (1ug) using NcoI. Digests were incubated 1hr @ 37C. Gel loading buffer was added to both samples and then run on a gel. Due to volume constraints, each sample was split and run in two lanes. Additionally, 0.5ug of undigested plasmid was run as a control.



Results:

external image 20101028.jpg

Gel Layout:
Lane 1 - Hyperladder
Lane 2 - Undigested 30 Hemat plasmid
Lane 3 - NcoI Digested 30 Hemat plasmid
Lane 4 - NcoI Digested 30 Hemat plasmid
Lane 5 - Hyperladder
Lane 6 - Undigested 40 Hemat plasmid
Lane 7 - NcoI Digested 40 Hemat plasmid

Lane 8 - NcoI Digested 40 Hemat plasmid




Samples were successfully linearized. Bands fromt the linearized plasmids were excised and purified using Ultrafree-DA columns (Millipore) according to manufacturer's protocol, spec'd on a NanoDrop1000.

Results:

external image 20101028%20DNA.JPG

Extremely poor yields and horrible 260/280 ratios. Samples were subjected to ethanol precipitation (0.1 vols of 3M NaOAc pH = 5.2, 2.5 vols of 100% EtOH, incubated @ -80C for 1hr, pelleted, washed with 75% EtOH). After EtOH precip, the samples were spec'd and there was essentially no DNA (data not shown). Will repeat the digests and use a different means for clean up, post digest.



20101022

Mini Preps - Hemat plasmid cultures from yesterday

Isolated plasmid DNA from 4mL of the 30uL and the 40uL using Qiagen's mini prep kit, according to protocol. Eluted each with 30uL of EB solution. Samples were spec'd on a NanoDrop 1000.


Results:

external image 20101022%20Hemat%20M.P.%20DNA.JPG

Great yields and great OD260/280 ratios. DNA will be digested with NcoI and gel purified next week.




20101021

Bacterial Cultures - Hemat plasmids 30uL and 40uL stocks

Inoculated 5mL of LB + 100ug/mL of Ampiciclin using a single colony from each plate from yesterday. Incubated O/N 200RPM, 37C.





20101020

Becteria - Hemat Plasmid 30ul and 40uL frozen stocks

Streaked frozen stocks (provided by Lisa) on LB/Amp100 plates and incubated O/N 37C.


qPCR - Hematadidium 40 DPP Std Curve for Validation Assay

qPCR on the 40 DPP standard curve made on 20101014. Master mix calcs are here. Plate layout/cycling params/etc. can be seen in the qPCR report (see Results).

Results:

qPCR Report is here (PDF).

Curve looks crappy, just like the 30 DPP curves from 20101008. Will prep fresh plasmid DNA and hope that helps.



20101014

Standard Curve - Hematadidium 40 DPP DNA

NanoDropped 40 DPP DNA from 8/18/2008 (provided by Lisa). Due to the poor appearance of the 30 DPP curves seen on 20101008, a new set of curves was made using a 1:10 dilution of 40 DPP DNA from 8/18/2008 (provided by Lisa). Calcs for the standard curve are here.

Results:

external image 20101013%20Hemat%2040%20DPP%20DNA.JPG


OD260/280 looks a bit rough. Will discuss with Lisa. However, this is likely due to the fact that this DNA was not cleaned up post-digestion which would contribute protein (enzyme and BSA) to the DNA sample, thus lowering its OD260/280.



20101008

qPCR - Check Hematadidium Standard Curves #1 and #2

Performed qPCR on 2 of 3 standard curves created yesterday. The third curve will be tested by Lisa, per the requirements for this validation. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF).

The curves look absolutely horrible and inconsistent. Not sure why. Maybe this DNA prep is too old? Seems unlikely. Will talk to Lisa and see if she has another stock of plasmid available.




20101007

Standard Curves - Hematadidium Std Curves for Validation Assay


Made 3 standard curves using Low TE Buffer. The curves were all the same and started with 3 separate stocks of "Dilute 30 DPP" from 8/18/2008 (provided by Lisa). Std curve copy numbers ranged from 3x10^7 - 3x10^1 (10-fold dilutions), 10, 3, and 1 copies. Will test curves according to validation requirements.



20100929

qPCR - V.parahaemolyticus Standard Curve Set Up

Used Qiagen extractions provided by Robyn that correspond to known CFUs of F11-3A strain. The CFU standard curve consisted of 10, 10-fold dilutions starting @ 2.4x10^9 CFUs. Here's the work-up sheet provided by Robyn. I also ran a dilution series of F11-3A that had been InstaGene'd by Robyn on 20090922 as a positive control. Finally, I made fresh working stocks of both probes. Mater mix calcs are here. Plate layout/cycling params/etc. are in the qPCR report (see Results).

Results:
qPCR report (PDF).



20100923

qPCR - V.parahaemalyticus Control Strain FH11-3A

Compared three different recipes for the Vp multiplex qPCR, looking at tdh and tlh: Nordstrom et al, 2007, Anne Baxter's, and mine. Master mix calcs and recipes are here. Plate layout/cycling params/etc. can be seen in the Results (see below).

Results:
qPCR report (PDF). Note: When viewing the qPCR Report, it should be noted that the Standard Curve graph cannot be used due to the fact that there are three different reactions labeled as Standard Curves on the plate. The software cannot (and does not) differentiate between these. Thus, the efficiencies calculated are for the combination of all three reactions and, thus, are not realistic. In order to see the efficiency for each different reaction recipe, one will have to manipulate the original data file (found on BackupOrDie) in the BioRad CFX Manager Software.

Interestingly, there's virtually no difference in detection of tlh (Texas Red) between all three master mixes. However, there is a significant reduction in sensitivity for the detection of tdh (FAM) in the Nordstrom et al, 2007 master mix, compared to the other two master mixes (which produce virtually identical Cts). So, this means I will use either Anne's or my master mix in future qPCRs for tlh/tdh detection.

However, there is something that seems a bit odd about this assay that I feel needs to be investigated. This strain has only a single copy of tlh. Knowing that, I would expect the same level (i.e. Ct) of tdh detection (or more, if there are multiple copies) as tlh. I've double-checked to make sure the probes are the correct sequence (i.e. names on stock tubes hadn't been switched). Next run I will create fresh working stocks just to eliminate the possibility that I mislabeled tubes when I made the original working stocks.






20100922

qPCR - Hematodinium Assay Specificity

Performed qPCR on a set of samples to assess the specificity of the Hematodinium assay. Master mix calcs are here. See qPCR report (in Results) for plate layout/cycling params/etc. Used the old standards set and the Hemat_191/214 probe.

Results:
qPCR report (PDF).

No detection in any of the non-hematodinium samples. Additionally, the standard curve looks fabulous. Will let Lisa know the results and find out where to go from here.


20100921

qPCR - Hematodinium Standards Comparison with Hemat_191/214 probe

Performed qPCR on old and new standards with the Hemat_191/214 probe.

Results:


qPCR - Hematodinium Assay Specificity

Performed qPCR on a set of samples to assess the specificity of the Hematodinium assay. Master mix calcs are here. See qPCR report (in Results) for plate layout/cycling params/etc.


Results:
qPCR report is here (PDF).

No detection in any of the non-hematodinium samples. However, the standard curve samples did not look good. Nearly a 10 Ct difference between the 1st and 2nd dilutions. As these are simply 10-fold dilutions, there should only be a ~3-fold difference between the two samples. The curve was the same used on 20100916 when checking the hematodinium probes.

Due to oddness of the new standard curve set that I made, will compare the old standards set with my new standards set using this probe (Hemat_191/214) to see if that makes a difference.



20100916


qPCR - Hematodinium Probes Test

qPCR was performed to find which probe (of the 3 provided by Lisa) works the best, as the probe used in yesterday's qPCR produced sub-par results. Compared Hemat_p and Hemat_191/214 probes. Master mix calcs are here. qPCR report (see Results) contains plate layout/cycling params/etc.

Results:
qPCR report is here (PDF).

The amplification plots are significantly better looking for both probes than the plots seen using the Hemat_p214. However, the dilution series isn't that great as there are a couple of instances where there's more than 3 Cts (10-fold dilution) between some of the standards. Additionally, the sensitivity seems to have decreased when compared with the Hemat_p214 probe.

It's interesting to also note that the efficiency calcuated by the BioRad software is poor (69%). However, when I process the raw fluorescence through PCR Miner, the gene efficiency is calculated to be 95% (PCR Miner Calcs are here). This fact lends credence to my thoughts that the BioRad software is only calculating efficiency based on Cts of the standard curve. If this is indeed the case, then a poorly made dilution series will have a negative impact on one's efficiency, as is seen here.



20100914

qPCR - Hematodinium Standard Curves Test

qPCR was performed using two sets of standard curves. One set is an old set created by Lisa. Tubes are labeled 1B-8B (3x10^7 - 10 copies) and are dated 8/19/08. The second set is a new dilution series made by me which mirrors the set from Lisa. Labels are 1A Hemat DPP - 8A Hemat DPP.

The new dilution series was made from a stock of Hematodinium plasmid DNA (30 Dilute DPP; 30 Dilute Digest Pure Hemat Plasmid 8.18.08). DNA was spec'd on a NanoDrop 1000 six times, an average concentration was calculated and used in calculating appropriate quantities of the plasmid/copy #.

Master mix set up is here. Plate layout, cycling params can be found in the qPCR report in the "Results" section below.

This qPCR used the Hemat_p214 probe. This was one of 3 probes available for this qPCR assay.


Results:
qPCR report is here (PDF).

The sensitivity appears to be great, however the amplification plots look atrocious. Oddly, the BioRad software calculates efficiency as being ~99%! I think the BioRad software calculates efficiency based on the standard curve and NOT on how the individual changes in fluorescence from one cycle to the next relate to expected changes in fluorescence from cycle to cycle. I need to look in to this in order to determine exactly how the BioRad determines efficiency.

The new standard curve set that I made seems to show an inaccurate dilution series, with more than 3 Cts (10-fold dilution) between some sets.

The old standard curve has some very odd discrepancies that make me question it's usabililty.

Possibly these problems observed with the curve (amplifcation plots and dilution discrepancies) are related to this probe? Lisa mentioned that one of the probes was no good, but she couldn't recall which one was bad, nor which of the three (if any) were good.

Due to the poor appearance of the amplification plots, I will repeat this qPCR and use try out the other two probes for comparison.






20100818

qPCR - Vp DNA stability test

qPCR was performed on samples spiked with Vp DNA stored in different fashions. qPCR conditions were based on results of a temp/Mg2+ gradients run 20100727, to optimize performance of the tlh and tdh primer/probe sets due to low efficiencies observed using the parameters found in Nordstrom et al., 2007. PCR for detection of V. parahaemalyticus in oysters. Appl. Envi. Micro.

Master mix calcs are here, using Bioline Immomix 2x Master Mix and FDA24/48 gDNA mixture. Cycling parameters/plate layout/etc. can be found in the qPCR report in the results section below.

Group layout of the plate:
5.27.10
6.3.10
6.17.10



Results:

qPCR Report (PDF).


Interestingly, according to the BioRad CFX96 and the positive controls that I used as standards, the efficiencies are lower than expected, despite the temp/[Mg2+] optimization that I determined 20100727. tdh (FAM) efficiency is show as 87.3%, which is acceptable. However, tlh (Texas Red) efficiency is only 65.2%.

Looking at an independent calculation of efficiency for both probes, I turned to PCR Miner and ran the raw fluorescence (no baseline subtraction!). The results can be seen at the bottom of these two spread sheets. When run through PCR Miner, tdh (FAM) efficiency is calculated at 80% and tlh (Texas Red) at 85%.

Personally, I find the numbers produced by PCR Miner to be more believable, primarily because just simply viewing the curves (see the qPCR report above) suggests that no samples have such low efficiency for Texas Red as calculated by the BioRad CFX96 (65.2%).

However, I'm still not totally thrilled by these results, as they also demonstrate that even in samples where equally quantities of positive control DNA were used (the samples used for standards), the Ct values generated from two primer/probe sets is not equal (tlh comes up significantly earlier than tdh). In fact, there's ~6 cycle difference! Currently, I'm not sure how this can be explained. I've spoken to Robyn to see if she has any knowledge on the V.parahaemalyticus genome and if either of these target genes exist in multiple copies, but she's not sure. I've contacted Anne Baxter in hope of shedding some light on this type of info as well as some input on this assay (thanks Robyn!).






20100727

qPCR - Vp Mg2+/temp gradient

In an effort to improve upon the poor efficiencies with these primer/probe sets, set up a temp and Mg2+ gradient in hopes of determining a more optimal set of cycling conditions. Mg2+ gradient was from 5.0mM (concentration used in Nordstrom et al., 2007. PCR for detection of V. parahaemalyticus in oysters. Appl. Envi. Micro.) to 1.5mM, with 0.5mM steps. Temp gradient was from 50 - 65C. Gradient layout can be viewed in the qPCR reports (below).

Due to the large number of samples, two plates/runs were needed to accommodate all [Mg2+]. qPCR master mixes using Bioline Immomix Master Mix are here: master mixes-01 and master mixes-02.



Results:

Raw fluorescence values were run through PCR Miner to determine gene efficiencies at each temp/[Mg2+]. Raw fluorescence and PCR Miner data is here.

qPCR report 1 (PDF) and qPCR report 2 (PDF) can be found here, which show plate layout/temp gradient.

The optimal temp/[Mg2+] are 3.5mM @ 59.5C, which yielded efficiencies of 87% and 86% for tdh and tlh, respectively. These efficiencies are immense improvements than what was seen using the parameters in Nordstrom et al., 2007. PCR for detection of V. parahaemalyticus in oysters. Appl. Envi. Micro. I'm satisfied with these efficiencies, which also means we can continue to use Bioline Immomix Master Mix.




20100721

qPCR - Vp efficiency check

Testing qPCR efficiency with tdh/tlh primer/probe sets using Invitrogen's Platinum Taq, due to mediocre efficiencies seen with both primer/probe sets using Bioline Immomix. The authors of the PAPER use Invitrogen Platinum Taq and we're hoping switching to this reagent will improve efficiencies for both primer/probe sets.

external image 20100721%20Vp%20efficiency%20check%20master%20mix.JPG




Results:

Efficiencies for both primer/probe sets are both LOWER than with Bioline Immomix Master Mix! Will need to attempt optimization (temp/Mg2+ gradients) to see if better performance can be achieved...

qPCR report (PDF).



20100714

qPCR - Vt isolates test

After optimization (see 20100707), ideal annealing temp and [Mg2+] were identified as 55C and 2.0mM, respectively. Performed qPCR on variety of RE isolates for Elene. Master mix set up is listed below:

external image 20100714%20Vt%20qPCR%20master%20mix.JPG




Results:

qPCR looks good for all samples except RE68, which exhibits two peaks in the melting curve analysis. This suggests two different sized products being generated in the PCR. Will discuss with Elene to see if this was an expected result for this sample. All other samples show a single melting curve peak, all at the same temperature.

Full qPCR report can be viewed here (PDF), which includes cycling params, amplfication/melting curve plots, standard curve and efficiencies calcs.




20100707

qPCR - Vt primer (VtpA F/R) optimization

Optimizing vtpA primer set by running annealing temp gradient (55 - 65C), as well as Mg2+ gradient. Master mixes for each [Mg2+] are listed below:


20100707_Vt_primer_optimization_master_mixes.JPG

Cycling parameters are as follows:

95C - 10mins
40cycles of:
95C - 10s
55 - 65C gradient: 10s
72C - 15s

Melt curve followed amplification.


Results:

[Mg2+] of 2mM at 55C produced the best efficiency (~97%). Data is here. Of note is that [Mg2+} of 3.0mM @ 57C produced 100% efficiency. Based on the appearance of the melting curves, these conditions were NOT selected, despite exhibiting 100% efficiency. Compared to the melting curve of the former (2mM, 55C), the 3mM, 57C appeared to have a small "bump" at the left-hand side of the base of the melting curve. I've decided to use the 2mM, 55C conditions based on the smooth melting curve and high efficiency.



V.tubiashii samples from Elene

Spec'd the following DNAs received from Elene. There were two tubes with RE22 DNA in them, so I differentiated them by tube color, yellow or pink Will optimize qPCR with the vtpA primers before qPCR on these samples.

external image 20100707%20Vt%20DNA%20specs.JPG





qPCR - V. parahaemalyticus-spiked samples


Performed multiplex qPCR using the tdh and tlh primer/probe sets on a variety of samples spiked with Vp DNA, from Robyn, that had been stored via various means at various dates. After consulting with Robyn, she suggested just using a 1uL of template from all samples.

Standard curve samples were run in duplicate.

Master mix:


external image 20100707%20Vp%20master%20mix.JPG

Results:

A spreadsheet with the sample Ct(s) is here and was given to Robyn for further analysis. Many samples did not amplify.

Here is the qPCR report (PDF), which shows cycling params/plate layout/etc.

Not included in the above report are the sample dates. Here are links to three screen captures showing the plate layout, including the sample dates (highlighted in dark blue in each picture):

5.27.10 layout
6.3.10 layout
6.17.10 layout

The results from the standard curve show poor efficiency for the FAM-based probe (tdh - ~55%) and mediocre efficiency for the Texas Red-based probe (tlh - ~77%). After discussing these efficiencies with Robyn, we've agreed that we should switch the exact same reagents being used in the paper from which these probes were established (Nordstrom et al., 2007. PCR for detection of V. parahaemalyticus in oysters. Appl. Envi. Micro.). Invitrogen's Platinum Taq has subsequently been ordered. Will repeat this qPCR with the new polymerase once received.





20100629

qPCR - V.parahaemalyticus Multiplex Primer/Probes Test


Testing out primer/probe sets.

Spec'd V.p. DNA (isolated by Robyn using Instagene on 20100429) on NanoDrop 1000:


20100629_Vp_DNA.JPG

DNA looks good. Will create dilutions of each sample of 100ng/uL for use in this qPCR test.

qPCR was set up with three different DNAs:

FDA03 = non-Vp no amplification
FDA24 = tlh+ only
FDA48 = tlh+ and tdh+

Master mixes can be seen below. Each master mix was for one rxn with each DNA and two negative controls (water, no template).

Cycling parameters (taken from Nordstrom et al (2007) Applied Env. Micro):

95C -10mins
45 cycles of the following:
95C - 5sec
59C - 45sec

20100629_Vp_Master_Mixes.JPG


Results:

qPCRs look as they should. No amplification with the FDA03 DNA. FDA24 only amplified with the tlh and the tdh+tlh mixes. FDA48 only amplified with the tdh and the tdh+tlh mixes. Will let Robyn know.

qPCR report, with plate layout.