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How expensive is DNA sequencing for plants?

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Often in the plant community you'll find yourself in a discussion (often around cacti) involving naming and identification of various plants. The arc of the discussion usually starts with crude estimating at what a particular clone a plant is, then comes refining of the 'model' that makes up the characteristics of said plant, and from there any further discussion disintegrates into pseudo-scientific ambiguity.

Sometimes (if you're lucky!) the topic of DNA sequencing is used to short-circuit the downward spiral into taxonomic hell: "well, we can only really know which plant is which once they've been sequenced."

Why the hell isn't this DNA sequencing happening so we can stop wasting all this precious oxygen on arguments around "what is a macrogonus", or "this is what a pachanoid looks like"? Is it really so prohibitively expensive that nobody has taken it upon themselves to send some samples into a lab to get sequenced? Am I trivialises/generalises how difficult it actually is to do the DNA sequencing of a plant?

I am genuinely curious. What are the practical barriers to getting, say, the Trichocereus genus (yes im sticking to old nomenclature) sequenced so we can settle the argument once and for all when it comes to identification? Imagine that this genus has already been sequenced, how would laypeople in the plant community then use this information to correctly identify their plants?

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This paper shows how researchers DNA sequenced 162 plants belonging to the genus Echinopsis.

MOLECULAR PHYLOGENETICS OF ECHINOPSIS (CACTACEAE): POLYPHYLY AT ALL LEVELS AND CONVERGENT EVOLUTION OF POLLINATION MODES AND GROWTH FORMS

Premise of the study: In its current circumscription, Echinopsis with 100–150 species is one of the largest and morphologically
most diverse genera of Cactaceae. This diversity and an absence of correlated characters have resulted in numerous attempts to
subdivide Echinopsis into more homogeneous subgroups. To infer natural species groups in this alliance, we here provide
a plastid phylogeny and use it to infer changes in growth form, pollination mode, and ploidy level.
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My understanding of this is it's not the sampling/ sequencing which is the expensive part any more ( providing you have a competent person and suitable facility to provide a quality sample- even these are readily available these days )

The exxy bit is getting the sequence data analysed by someone who knows what they're doing. I know a few such people socially and once they started to explain the process to me my head assploded. Mind you last time I checked was like 2011

Once the day comes when you send in your plant sample and a machine spits the data back at you in a form you can understand ( Echinopsis- yes! ) then we'll be able to get all these tricky taxonomic questions sorted.

Til then just let the taxonomists flog it out in the carpark, they're a tetchy bunch, it can be hard to keep two of them in a room together ;)

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shotgun assembly is shit...... (ie putting a genome together usually gigabases together from 150base reads)

As darklight said it costs a lot to build a decent scaffold and get the contigs (long linear DNA) to a small number (closer to the number of chromosomes the better.)

Usually you start with a transcriptome (mRNA) to get a feel for expression and whats going on

Sometimes mitochondrial or chloroplast genome sequencing is a better idea.

You don't really need to have the whole genome, the paper mu! posted is not whole genome sequencing it is plastid or plastome sequencing which is just little dsDNA circles that provied sub functions in the cell like cpDNA (chloroplast) (which is a type of plastid)

This avoids the need do assembly as the short reads can be stitched together from previous assemblies (scaffolding) and its not that huge so you can run a few plastomes in parallel (how the paper got so many done) on the one illumina chip which is what the "cost" of sequencing is, other then the couple of 12hr+ days you need to do for library prep

if you had a few plants you wanted done (10+) and someone who was good at not fucking pipetting up or contaminating PCR reactions then you could do all of the plastomes for the cost of one "sequencing event" or you could make a snpchip that did it for specific lineages (cactus might be possible), which is based off nucleotides in the dna that change from plant to plant but doesn't really mean much in terms of function

Edited by Tripyamine
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If i had sequences for monstrose or crested genes, and primers for those genes, i could perform genotyping via PCR. 

Not sure about how to genotype for pach, bridgesii, peru, But i have expirence genotyping single and double mutant genes in arabidopsis thaliana.

 

So how are we gonna get these sequences, im getting sick of waiting, this needs to happen now. 

 

 

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The exxy bit is getting the sequence data analysed by someone who knows what they're doing. I know a few such people socially and once they started to explain the process to me my head assploded.

 

 

I was wrong. There are places in .au which will extract your DNA and run all kinds of cool tests for about $100. They can give you results in spreadsheet form which are easily understood by people with minimal experience. They can provide more help for more cost.

 

The problem is that you need  genetic *markers* for whatever it is you are seeking. These are similar to the controls you run in any experimental setup.

 

You need specific markers for your specific test. And if these markers don't already exist on some database, you need to develop them, and that's the exxy part.

 

For example, if you need to determine whether your Acacia is species x or species y, you will need the genetic markers for one or both of those species, so you can tell whether your sample fits the profile. If there are no markers on public record, you will need to track down the part of the genetic code which is consistently different in either species. Apparently this can be a PITA, time consuming and exxy.

 

However ( a separate example here ) if you need to determine whether your sample is a clone of Acacia x, or a seed grown individual, you will need different markers to those outlined in the example above. Again, if these markers do not exist on public record you will need to develop them.

 

Plant species such as tobacco, rice, wheat, and that bloody tedious Arabidopsis thaliana have plenty of markers on record, often available to the public. Is less likely that your obscure ethnobotanicals will have such records on them. There are places to check, but no idea where to find them. One place to start looking is formal published genetics studies on your species of interest. Another are the databases.

 

Got some sequencing done recently for a common species, there were markers on public record for it. All up the bill was cheap, with supplied analytical spreadsheet.

 

 

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Another example is drought tolerance within a species.
 

It may be known, for example, that the presence of certain genetic markers in that species has a high correlation with drought tolerance.

 

You can use those markers to screen a large population of that species, and select individuals to test for drought tolerance to breed from

 

etc

Edited by Darklight
correcting my shithouse grammar

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There are places in .au which will extract your DNA and run all kinds of cool tests for about $100. They can give you results in spreadsheet form which are easily understood by people with minimal experience. They can provide more help for more cost.

 

No need to pay for DNA extracting, ill do it for free 

My major concern is how to get it sequenced for as cheap as possible. 

 

Idealy id like to get the sequence for a dozen different trichos, then get the sequence for Nitro & Zellys TPM, TPQC aswell as TBM so i can start to map out which genes are mutated. Then i want to use CRISPR to create mutations in the candidate genes of wild type trichos, so i can grow out the genetic knockouts and write a research paper about the findings :)

 

Would you mind providing the .au websites your refering too?

Im very keen to see what sorts of testings can be performed at what costs. 

Edited by Change
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Sent you the link to the sequencing place Change. It's not a state secret, but dealing with them does require a degree of technical knowledge

 

CRISPR is for inserting and knocking out genes, not mutating them AFAIK. Happy to be corrected on this. Are you as technically up to speed on all this as you think you are?

 

Mate, if you are in the Hunter and are playing with CRISPR you prolly want to be working out of a registered PC2 facility. Not sure about knockouts and OGTR, but unless you want to make ugly front page news I'd do a thorough OGTR legislation check and print out and keep the relevant documents next to you laminated in case of incursion.

 

What you're proposing sounds like a PhD at the very least. More likely six PhDs.

 

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Thanks for sharing the info Darklight 

Yer maybe i didnt do the best job of wording my idea, Ill try again 

 

Using CRISPR you could insert or remove nucleotides in the promotor sequence of the candidate genes rendering them non functional.

These none fuctional promotor sequences would block the expression of the candidate genes so we could observe the results of this mutation in wild type trichos. 

I didnt mean mutagenesis using CRISPR. I ment specifically knocking out promotors so we could observe the effects of these specific mutations in trichos. 

 

Currently im doing some volunteering in a plant research lab that is performing very similar experiments with genes in arabidopsis thaliana, in an attempt to better understand there funtion. I certainly dont think im technically up to speed to perform this project by myself next week. But im alot closer than i was 1 year ago, and as every day passes my technical knowlegde expands. Im quite aware CRISPR needs to be performed in a registered PC2 facility. This isnt an at home project, its a academic project which id like to perform and publish with the approval of my supervisors.

 

But we dont need PC2 to start extracting the DNA and getting it sequenced. There is alot of work that needs to be performed before we can start the genetic transformations. If they let me do 6 PhDs on this project it will be my dream come true :)

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But we dont need PC2 to start extracting the DNA and getting it sequenced.

 

Completely true. Do all the groundwork while you can!

 

If they let me do 6 PhDs on this project it will be my dream come true

 

Mmm hmm... you say that now... ;)

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I agree with Darklight's comment "The exxy bit is getting the sequence data analysed by someone who knows what they're doing."

 

This is ongoing for Lophophora right now so I can greatly appreciate what that means. Interpreting the preliminary analysis was something like $8K and the necessary follow up including some additional sequencing went well over $20K. No human actually does this part as it is far to complex. Software does the number crunching and then a human interprets those results.

There is a surprising degree of capriciousness within this picture so a disturbing part of what has been published is really no more solid than a house of cards. The bar really needs to be raised a notch imho.

 

The cheap tests and results (like one would use to learn about their ancestry or potential medical conditions) involve identifications of known markers and are not actually sequencing anything. However, if sets of markers had been established for our plants of interest that sort of test could certainly be developed for this narrow area.

 

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I agree with Darklight's comment "The exxy bit is getting the sequence data analysed by someone who knows what they're doing."

 

This is ongoing for Lophophora right now so I can greatly appreciate what that means.


 

 

OMG omg omg Trucha, and thanks for sharing this. So excited you're doing this from scratch

 

What trucha is describing folks is the current gold standard for starting from scratch. Whole genome sequencing analysis. Then comes the development of marker sets. Two separate processes. There is a lot of overlap in common parlance, and even some scientists will take the linguistic shortcut.

 

There is a surprising degree of capriciousness within this picture so a disturbing part of what has been published is really no more solid than a house of cards. The bar really needs to be raised a notch imho.

 

Yep that blew me out. I was looking at whole genome sequencing of Ganoderma in 2014, talking to a couple of genetics post-docs and aside from the initial expense I was blown out that results can even differ depending on which brand machine you use. There is no one-size-fits-all standard, or wasn't at the time. And the blinkenlights machines are exxy and are superseded every few years. Whole genome sequencing is frequently outsourced even in mid-size facilities where the exxy toys of +5 years ago aren't up for replacement and yet almost obsolete

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Very interesting, thanks for joining the conversation trucha, Im not sure where your located, but if your ever passing through the hunter valley, please get it touch, id love to pick your brain for an hour or 3 :)

 

Have they published anything yet? 

If not when they do, could you please post it here 

 

 

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im going to enter into this conversation properly in the next day or so once my brain come back from holidays.

 

but whole genome sequencing is pretty much the future but it becomes complicated, because of the insane amount of data and then there is the processing.

 

There is a lot of guesswork and a lot of "winging it!"

 

but given i am entering into this field of study i should probably contribute

 

 

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Yes please Obtuse, kick in :D

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oh lordy, moving around fastq files is a right pain thank the lord for nectar and aarnet filestore 

Beijing genomics institute just/is opening up in aus so sequencing might get a little cheaper here. 

At the moment its costing me about 3k to do the sequencing on say 12 different "things". I do transcriptomics so for me its 6 different treatments of a yeast and a replicate. Im a bit lucky and we have the illumina, iontorrent and pacbio rigs in the lab upstairs so its just the painstaking mRNA extraction and then library prep but, at that point it gets given to a tech and the magic happens. My research group (if its important) doesn't have to pay for sequencing, however, we have to pay for the kits and the flow cell for the machine so thats what the 3k is. 

Im lucky and can handle most of the bioinformatics myself so no need to pay for any of that which can get very exxy and ive seen it too many times in the past that they just flat out balls up the analysis and you get bad results, that should be fine but nobody ever double checks because they are 20gb and take a day to crunch at the least. 

It should be interesting to see how pacbio and nanopore drop the price, I cant wait to run something on a pacbio2 long reads is where its at for large plant genomes so much repeat that make it cooked to assemble.

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I don't understand about 80% of the technical speak of the above, and genetics isn't my field

 

Am incredibly impressed tho, by how much the skillsets of youse all have progressed :)

 

It's been nearly 3 years since the OP put up their request. What's changed?

 

My understanding is that SDN-1 knockouts are now no longer covered by OGTR legislation, but not sure if this covers environmental release as well. Anyone know?

 

Quite possibly what hasn't changed, and may be going backwards, is public access to a lot of new databases.

 

And I suspect ( but cannot confirm ) that the human factor is still a big part- quality DNA extraction is hard for some species or plant parts and as Tripyamine says above, a bad result needs to be caught before it proliferates. Add that to the variables of technique and machine and it sounds like it'll be a shitfight for a while yet

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Well everything is cheaper. Much cheaper. And extractions of most non-agricultural crops produce very high quality reads now (a few years ago it was mostly crops that produced high quality reads).

 

It's still difficult to predict what species will work though. Even between different populations of some species you can get chemotypes that won't extract properly from one method or another. If one extraction method fails, try another. Always sample the very tip of clean, healthy new growth to ensure you don't sequence anything you shouldn't. Also, new growth has higher cell density and since each cell has three sets of DNA, you've got a higher DNA  to other material ratio which helps with extraction.

 

I'm assuming you want to mess with genes on the nuclear DNA? Keep in mind you've also got chloroplast and mitochondrial DNA too. They're generally more useful for long term lineage resolution.

 

 

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Ive gone off the DNA sequencing idea, mostly because it doesn’t answer the questions im asking in the first place and would end up being an expensive experiment that doesn’t help with much more other than IDing plants or trying to trace back their evolutionary origins (both of which could be decades of work). A few years back I was really interested in attempting to work out the underlying mechanisms causing funky phenotypes in cactus, for example TBMs and Nitrogen hybrids. But these days I realise the answer to these questions is unlikely to be found via DNA sequencing and instead would require RNA sequencing. Furthermore my focus has changed from external to internal, so rather than caring about what is causing phenotypes, im now more fascinated with understanding metabolism, and metabolite accumulation and the best way to study metabolism is RNA sequencing because it provides direct evidence as to what genes are actively being expressed, as opposed to DNA sequencing which tells you what exist within the genome but gives no indication of expression levels.

 

Currently we have no evidence to suggest that mutant cactus phenotypes are being caused by mutated genes, and its just as likely that none of the metabolic genes are mutated but instead sections of regulatory DNA (promotors / enhancers/ or silencers) or perhaps transcription factor binding sites and or sRNA binding sites might be mutated, causing dysregulated gene expression, resulting in mutated phenotypes. Therefore, to gain a better understanding of whats going on, RNA sequencing from both wild type and mutant plants would provide useful information into the differences between gene expression within the two groups of plants. One of my colleagues just had 12 Arabidopsis plants (6 mutants, 6 wild type) RNA sequenced by AGRF and the cost was approximately 10k. Sadly without some wealthy backers to fund my research project this will probably just remain another idea on the list of things I wish I could afford to do.

 

I cant comment on SDN-1 knockouts as I have no experience working with them. My research group uses SALK Institute knockout lines of Arabidopsis (T-DNA insertional mutants), as well as the Agrobacterium mediated transformation method to create transgenic populations. Both the mutants and the transgenic plants require PC2 facility and must remain within the quarantine zone at all times. It would be fantastic if we could use SDN-1 to make the same mutants and grow them in uncertified glass houses, as recently the reproductive group gained approval to demolish several million dollars’ worth of PC2 glass houses which they are in the process of replacing with a mice testing facility (PETA protestors welcomed). This has significantly reduced the space available for plant research as well as prevented us from working with species that are to large to fit inside growth cabinets. Its crazy to think reproductive research is receiving higher priority compared to plant research, when we already have an overpopulation problem, and the current projections suggest agricultural yield trends will be insufficient to feed our growing population by 2050. But this is all a result of Australian university’s shifting there focus to industry funded research with the goal of increasing profits rather than fundamental research with the goal of expanding knowledge.

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Not much has really changed in the space, Illumina has a bigger machine and nanopore has released some new flow cells that are making Illumina and Pacbio quiver a bit methinks.

I did a few algae genomes last year for $2k with nanopore kit, it's still a bit of a pain in Australia because there are no distributors so you need a specific Bicon permit and the shipping is killer. But long reads are amazing, just not accurate enough but great for resolving repeat sections in genomes, Illumina is still kinda unavoidable. 

 

SDN-1 is kinda funny, it has to be markerless to start with. From what I understand its no plasmid and no DNA. That means you need ribonucleoproteins ie the pure cas9 protein and a pure guide RNA that will randomly perform insertion or deletion and you don't even really know how many bases will get removed or inserted because it depends on what repair mechanisms are available in the organism you want to transform. This gets really tricky because you have only really one option, you have to fuck with a gene that will allow you to select a line that has your SDN-1 as well, this means it probably won't have it and just your selection in/del will be present. So there is still a lot of parallel replicates needed in order to get a line with what you actually want. 

You cant insert a gene, you can only remove its function or slightly modifies it. 

 

And yeah @Darklight the hardest thing about nanopore and Pacbio is the extraction, you need ultra-pure and unsheared DNA if you want to get long reads and the most out of your flowcell.

Kinda funny but, there is a legend from ANU that has made a group on a website called protocols.io that has customized extractions people have perfected for heaps of different tissue. 

It took me the better part of 4 months to get my extractions clean and big/unsheared enough to run, I had to make up a whole custom protocol.  

Database access is still amazing, bioinformatics is mostly opensource still, between GitHub/NCBI/jgi/ebi basically everything is there, not everybody does it but you should put your raw data on the sequencing read archive (SRA) at the NCBI. So you can go on SRA make an account and reassemble peoples work with newer/better techniques/use it to beef your own sequencing up. 

 

Something I have been doing a lot of is metagenomics, which is heaps of fun. Take a sample with a complex mixture of organisms and sequence everything and see what's in the pool. Its a really cool diagnostic tool for anything from gut microbes to algae in a creek. I think Metagenomics is something lots of you guys might be interested in because its a way to look for pathogens (bacteria/fungi/virus/etc) with plants and is starting to get really approachable price-wise. I can do a 16/18s run on Illumina for ~$80 (but that's just prok/euk) and if you want to look at everything you just extract DNA and run it on a nanopore and blast as your sequencing, start to see everything in realtime which is cool for viruses and weird stuff. 

 

 

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8 hours ago, Tripyamine said:

@Darklight

 

Kinda funny but, there is a legend from ANU that has made a group on a website called protocols.io that has customized extractions people have perfected for heaps of different tissue.

 

Very cool...

 

"Scientists are constantly re-discovering knowledge that others have not had the time to publish and improving existing methods without the ability to share the improvements. Our mission is to change this with a free, up-to-date, crowd-sourced protocol repository for researchers."

 

http://www.protocols.io

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On 20/08/2019 at 10:39 AM, Tripyamine said:

Kinda funny but, there is a legend from ANU that has made a group on a website called protocols.io that has customized extractions people have perfected for heaps of different tissue.

 

 

If it's the same legend from ANU who was proposing exactly that a few years back, please tell him I said hi? He was a legend even back then, now I reckon he'd be a fucken rockstar

 

Great site too

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Right now what I'm doing is drooling. I don't understand 80% of the techspeak, which is entirely normal for a new field for me, so I'm not bothered by that. If I needed something doing in this space right now I'd spend the time because obvs the field is expanding and I need to watch the directions. All I can do at this point is observe, and drool

 

What excites me is that there are people here with relevant and current skillset who can explain it well and who are sharing information

 

Would it be possible for 2-3 of you to get together and put something up for the next EGA Garden States? Like a DNA 101 workshop on a single relevant point to us ethno freaks?

 

A few of us have been working on practical protocols for base-level workshops for the day next May. Light microscopy, a little TLC, and something fungal again. Stuff people want to do at home- and can- but just need a few pointers to get the most out of the tek

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