I had my water tested but need explanation

[User1]

Generally, for each of the PCRs, you would run "blanks" or negative controls. If they amplify DNA, then you know that there was cross-contamination in the DNA extraction or PCR prep. You wouldn't proceed to the DNA sequencing step if you had contamination.

As for step 7, the DNA sequencing, I'm not sure what they do at the facility. Since these facilities run 1,000s of samples daily for research labs around the country, any problems would be reported very quickly. Unlike the ICP labs that do water tests for hobbyists, labs that do DNA sequencing for researchers must be 100% transparent about their methods.

Got it, thanks.

 

[AquaBiomics]

Got it, thanks.

Yeah, lexinverts covered the fundamental answer: blanks (negative controls). I'll add a couple details specific to these reactions.

Like most researchers in the microbiome field, I include blanks for two levels

1. No-template controls - run the reaction with just water. A positive result would indicate contamination and require redoing the reactions with new reagents. For this reason, molecular biologists take great pains to avoid contamination, because it costs time and money when it happens.
2. Extraction controls - because some microbiologists study samples with just a few cells (literally unmeasurable amounts of DNA using most methods), they noticed that many DNA extraction kits contain bacterial DNA. So a convention developed to also include "extraction blanks", simply tubes of pure water treated exactly as if they were real samples (filtered, extracted, PCR, etc). This controls for any bacterial DNA that might be present in the kits.

Both of these controls always come up empty during PCR while samples show clear bands. But only because I am trying hard to prevent contamination, it is always a moment of relief when they show up clean.

In the kind of sequencing run we are using (Illumina MiSeq), my batch of samples is sequenced without samples from any other users on the machine. So I don't have real concerns about contamination with other samples. And I label each clients samples with a unique combination of synthetic DNA "barcodes", so each DNA molecule loaded onto the instrument is physically labeled with the sample from which it came.

Nothing's perfect, everyone can make mistakes, but the procedures developed by the community of biologists using high-throughput DNA sequencing are pretty effective at preventing this errors and detecting them if they happen.

 

[User1]

Yeah, lexinverts covered the fundamental answer: blanks (negative controls). I'll add a couple details specific to these reactions.

Like most researchers in the microbiome field, I include blanks for two levels

1. No-template controls - run the reaction with just water. A positive result would indicate contamination and require redoing the reactions with new reagents. For this reason, molecular biologists take great pains to avoid contamination, because it costs time and money when it happens.
2. Extraction controls - because some microbiologists study samples with just a few cells (literally unmeasurable amounts of DNA using most methods), they noticed that many DNA extraction kits contain bacterial DNA. So a convention developed to also include "extraction blanks", simply tubes of pure water treated exactly as if they were real samples (filtered, extracted, PCR, etc). This controls for any bacterial DNA that might be present in the kits.

Both of these controls always come up empty during PCR while samples show clear bands. But only because I am trying hard to prevent contamination, it is always a moment of relief when they show up clean.

In the kind of sequencing run we are using (Illumina MiSeq), my batch of samples is sequenced without samples from any other users on the machine. So I don't have real concerns about contamination with other samples. And I label each clients samples with a unique combination of synthetic DNA "barcodes", so each DNA molecule loaded onto the instrument is physically labeled with the sample from which it came.

Nothing's perfect, everyone can make mistakes, but the procedures developed by the community of biologists using high-throughput DNA sequencing are pretty effective at preventing this errors and detecting them if they happen.

Thank you for the information and explanation. I appreciate it.

It would be safe to say than this isn't a replacement for the ICP test(s) nor something most hobbyist would use. From a cost perspective not data value.

I also thought it was interesting in how we put an age on a tank. This isn't an argument but more of a thought or question again related to data. If we move a mature tank or add to it how does that affect the data. I personally view a move as a storm crashing through a reef crest and new sand, debris, and die off is going to happen. So maybe it is a moot point.

Just looking at it from a data stand point and how you catalog it.

 

[chefjpaul]

Paul, just made the discussion of this technology more interesting.

Very complex topic that definitely will always have an infinite variability, but definitely one of my peak interests. This will be enjoyable to watch evolve.

*side note- I hope Eli can take a passion, that he is sharing with us, and make a fortune doing so. Few get that opportunity.

 

[Paul B]

If we move a mature tank or add to it how does that affect the data. I personally view a move as a storm crashing through a reef crest and new sand, debris, and die off is going to happen. So maybe it is a moot point.

I moved this tank here 60 miles last year. I didn't lose anything, not even a pod.

 

[User1]

I moved this tank here 60 miles last year. I didn't lose anything, not even a pod.

Yep, I remember. I've also moved a tank although 22 miles, not 60 I was looking at it more as it relates to the water test, or any test for that matter, and is that considered a break in the timeline because of it. Larger tank, smaller tank, add to such as rock, substrate, or even die off. Maybe it isn't a big deal and we treat it like mother nature throwing a storm at it.

 

[AquaBiomics]

I also thought it was interesting in how we put an age on a tank. This isn't an argument but more of a thought or question again related to data. If we move a mature tank or add to it how does that affect the data. I personally view a move as a storm crashing through a reef crest and new sand, debris, and die off is going to happen. So maybe it is a moot point.

Just looking at it from a data stand point and how you catalog it.

I've wondered the same thing. I can see arguments for considering it a partial restart when we move existing rocks, water, sand, etc into a new tank, whether its just a tank upgrade or a move across country. On the other hand, its not clear what kind of "age penalty" should be applied in such cases. I've concluded chronological age is probably the best we can do.

With that said, I'm sure a tank move or tank upgrade affects the microbiome. I say this because its been shown that even a large water change affects the microbiome, increasing diversity of the community. Doesn't mean I'd consider it a reset to time=zero but the microbiome is sensitive and dynamic...

 

[chefjpaul]

I've wondered the same thing. I can see arguments for considering it a partial restart when we move existing rocks, water, sand, etc into a new tank, whether its just a tank upgrade or a move across country. On the other hand, its not clear what kind of "age penalty" should be applied in such cases. I've concluded chronological age is probably the best we can do.

With that said, I'm sure a tank move or tank upgrade affects the microbiome. I say this because its been shown that even a large water change affects the microbiome, increasing diversity of the community. Doesn't mean I'd consider it a reset to time=zero but the microbiome is sensitive and dynamic...

Going back to your live rock theory though, if the microbiome are sensitive yet dynamic, then assumptions could be repopulate. How, though, say the exact system, maintenance, and habits are kept?

I mean the tank itself is just the box anyway.

 

[Paul B]

Yep, I remember. I've also moved a tank although 22 miles, not 60

If I only had to move 22 miles, I would have made my fish walk there.

The move was a big deal for my tank I imagine even though I moved everything in vats including the water. Most of the mud from under the RUGF had to be lost but I wanted to start new mud anyway and it almost clogged the space under my UG filter.

Here my Son n Law is lifting the filter plate for the move. You can see a lot of mud that accumulated over decades. That mud was full of life but I couldn't see putting it all in a new tank so I only added a little of it. Then I collected some new mud.

The new mud came from a different part of Long Island so I am sure the biome was different.
My rocks were not cleaned so a lot of that life was saved and my gravel was only rinsed in old seawater so much of that was also saved.

The only difference I can see in the tank now is that it seems even healthier and SPS corals grow well. But I also have much brighter lights as my old DIY water cooled lights croaked here with the high humidity when I moved in. I am also 100 yards from the sea so the air is much more humid.

I am not sure if the new conditions did this but my blue sponge is growing much faster here. Probably three times as fast. That may be due to the NSW I use here or the lighting. Probably a little of both.

In the spring I will again add some rich mud that I will have to find in a lagoon on the north shore of Long Island as here it is mostly sand.

 

[ScottR]

I read your report. You need more lectrolytes, mmkay.

 

[brandon429]

Paul at that range to the ocean your tank is actively exchanging microbiome inhabitants by the hour via aerosol vectoring (especially during storms/upsets) that’s pretty neat. We get activity via the air at that distance...not just direct exchange. Algae, planktors, bac

It will be interesting to see how bac measures relate in another couple years given the activity, how the distributions hold up over time / selections within your tank etc

 

[Reesj]

AquaBiomics tested my water a few weeks ago and I am all excited to know what it means as I can't interpret most of it so I need Randy who has more degrees than a thermometer, and I am an electrician with a fish tank.

Years ago I couldn't spell Lectrician but now I are one.

Thanks for doing this. Really helps the rest of us to get an idea about microbeals in established tanks , Tagging along..

 

[lexinverts]

This article covers some of the topics discussed in this thread:

Establishing a Healthy Microbiome in a New Aquarium Using Live Rock

Studying the effects of live rock in a newly established aquarium shows that high-quality live rock promotes the rapid establishment of an effective biological filter and a microbial community similar to those found in mature reef tanks.

www.reef2reef.com

 

[klp]

Thankyou Paul! I did not know this service existed. Thankyou AquaBiomics for providing a very interesting service. I would love to see you test some of the readily available bacteria in a bottle many use to start their tanks AND see how they survive during a three to six month period. Test against a tank that is left to go through the cycle without any bacteria added. Maybe a good time to team up with BRS for testing? Just a thought but one that I think many would be interested in. Thanks again to Paul B and AquaBiomics for this very interesting discussion and information.

 

[Paul B]

I think it is an excellent service and very interesting.

 

[User1]

I think it is an excellent service and very interesting.

I'll give it a go. Mil discount? Starving parents (2 in college)? Will wash your car

Just kidding. I was visiting the site last night to see what made sense. Put something on my Christmas list - may make a good stocking gift.

 

[AquaBiomics]

Thankyou Paul! I did not know this service existed. Thankyou AquaBiomics for providing a very interesting service. I would love to see you test some of the readily available bacteria in a bottle many use to start their tanks AND see how they survive during a three to six month period. Test against a tank that is left to go through the cycle without any bacteria added. Maybe a good time to team up with BRS for testing? Just a thought but one that I think many would be interested in. Thanks again to Paul B and AquaBiomics for this very interesting discussion and information.

Yeah, the bacteria in a bottle question is hard to avoid.. I have actually done one of these experiments already, and will write it up soon. Duplicate dry rock vs dry rock + bottled bacteria tanks, side by side, for a month. I just didnt add it to the live rock vs dry rock article because it was a monster already!

I think I can say without ruffling any feathers that teaming up with vendors or manufacturers of these products presents some special issues to be considered. On the other hand, if hobbyists themselves conduct the experiments and I just provide an independent service testing the results, these challenges could be avoided.

So I hope to support lots of testing of these products by hobbyists, and will do more of them myself as resources permit... there are a lot of products out there, and my capacity for DNA analysis is much higher than my capacity for tank experiments.

[edit - removed a sentence fragment missed during earlier edits]

 

[Back where it all began]

So the questions become, is all diversity equal, how do you determine what is lacking in your tank and how is the best method to add this agent if indeed it is needed for your tank based upon these results.
This is a great study, but cause and effect are extremely complicated subjects to interpret and may take a long time to link together.

 

[Oldsalt]

I'm in awe reading this thread. I was born and raised in Queens NY but never had a marine tank unt I emigrated to Perth Western Australia in 1983. I only ever used 100% NSW that is collected from the Indian Ocean which is filtered through >500 micron, allowing all but the largest plankton through. The water is routinely collected from a depth of ~ 20 feet which is approximately another 20 feet above the substrate. Importantly, the water is always collected well off shore by ~ 3 km (>1 mile) and well upstream of any known pullution sources - ie., sewage drains, chemical processing plants and coal powered electricity plants.

This part of my post is directed to @AquaBiomics:
I'd be very interested in getting a sample of my water analysed by you, however, would a sample sent to you from such a large distance away degrade, skewing the results? If not, how do I arrange a delivery to you?

 

[AquaBiomics]

So the questions become, is all diversity equal, how do you determine what is lacking in your tank and how is the best method to add this agent if indeed it is needed for your tank based upon these results.
This is a great study, but cause and effect are extremely complicated subjects to interpret and may take a long time to link together.

You bring up important questions, and I think we all agree we're in the early days of our efforts to answer them.

Is all diversity equal, I suspect not.. for one example, in my live rock study the diversity of the two different live rock sources was not very different but their effects (and nitrifying microbe contents) were quite different.

Diversity isnt the only way to look at these communities. I like to also ask, how similar are the levels of each type to their levels in the typical reef tank? And what are the levels of particular types like nitrifying bacteria or pathogens? All of these can vary independently from diversity.

I don't think diversity per se is necessarily the ultimate goal. The goal in my mind is a stable microbial community that provides a range of different services to maintain a healthy ecosystem in the aquarium. Diversity is one descriptive statistic that an aspect of this complex system. Microbial ecologists consider diversity a useful statistic, but its not the only thing to consider.