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- Transposon insertion sequencing (Tn-seq) combines transposon insertional mutagenesis with massively parallel sequencing (MPS) of the transposon insertion sites to identify genes contributing to a function of interest in bacteria. The method was originally established by concurrent work in four laboratories under the acronyms HITS, INSeq, TraDIS, and Tn-Seq. Numerous variations have been subsequently developed and applied to diverse biological systems. Collectively, the methods are often termed Tn-Seq as they all involve monitoring the fitness of transposon insertion mutants via DNA sequencing approaches. Transposons are highly regulated, discrete DNA segments that can relocate within the genome. They are universal and are found in Eubacteria, Archaea, and Eukarya, including humans. Transposons have a large influence on gene expression and can be used to determine gene function. In fact, when a transposon inserts itself in a gene, the gene's function will be disrupted. Because of that property, transposons have been manipulated for use in insertional mutagenesis. The development of microbial genome sequencing was a major advance for the use of transposon mutagenesis. The function affected by a transposon insertion could be linked to the disrupted gene by sequencing the genome to locate the transposon insertion site. Massively parallel sequencing allows simultaneous sequencing of transposon insertion sites in large mixtures of different mutants. Therefore, genome-wide analysis is feasible if transposons are positioned throughout the genome in a mutant collection. Transposon sequencing requires the creation of a transposon insertion library, which will contain a group of mutants that collectively have transposon insertions in all non-essential genes. The library is grown under an experimental condition of interest. Mutants with transposons inserted in genes required for growth under the test condition will diminish in frequency from the population. To identify mutants being lost, genomic sequences adjacent to the transposon ends are amplified by PCR and sequenced by MPS to determine the location and abundance of each insertion mutation. The importance of each gene for growth under the test condition is determined by comparing the abundance of each mutant before and after growth under the condition being examined. Tn-seq is useful for both the study of a single gene's fitness as well as gene interactions Signature–tagged mutagenesis (STM) is an older technique that also involves pooling transposon insertion mutants to determine the importance of the disrupted genes under selective growth conditions. High-throughput versions of STM use genomic microarrays, which are less accurate and have a lower dynamic range than massively-parallel sequencing. With the invention of next generation sequencing, genomic data became increasingly available. However, despite the increase in genomic data, our knowledge of gene function remains the limiting factor in our understanding of the role genes play. Therefore, a need for a high throughput approach to study genotype–phenotype relationships like Tn-seq was necessary. (en)
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- "Product"? Product of what? Something I can buy on Amazon? Presumably they mean the product of some chemical reaction, and presumably the PCR chemical reaction, but it's not clear, and if that's really what is meant, then why not say "the PCR-amplified sequences" (en)
- does "Mariner" refer to the Himar I Mariner transposon? also it says recognition sites are introduced somewhere, introduced where? what are recognition sites? why are they introduced and why do we care that they are introduced? (en)
- What is a "staggered cut" as opposed to a "regular cut"? Does it have something to do with "cleaving"? I thought we wanted to cleave something 4 base pairs before the end of the terminal repeat. Now we are doing something with a recognition site somewhere? (en)
- I know what this means but there has to be a less jargon-y way of saying it. DNA sequences are not literally streams or other flowing bodies of water, and the layperson shouldn't be expected to be familiar with the analogy (en)
- I just realized I don't even know what is meant by "recognition sites" here or why I should care about what they are or what they have to do with anything (en)
- Is DNA shearing the same as DNA cleaving and DNA cutting and "restriction"? Could this article just please choose one simple and non-jargony term and then stick with it throughout? (en)
- What on earth does it mean for a genetic interaction to be aggravating or alleviating? Do genes suddenly now have feelings? And are genetic interactions supposed to be understood to mean the same thing as gene interactions? If so, why not use the same term throughout the article? (en)
- What does ligation mean? Probably unnecessary super jargon-y term. At the very least link to an article explaining it (en)
- presumably this means "cut", since that's what it says in the diagram, but I'm not sure about the jargon (en)
- Again, more jargon, what is an adaptor, I hear it all the time but it does not make any sense to me and seems to be another unnecessarily complicating and obfuscating term (en)
- What on earth is a "base overhang"? Why should we care? Does it matter for understanding the method whether there is anything facilitating anything? Or is that just a random organic chemistry fact that will be irrelevant in future iterations/versions of this overarching method? Why should the reader care (en)
- I am not even going to bother to pretend to understand what this acronym refers to. Googling "page" will almost certainly not lead to any useful results. Again a Wikipedia link or some other external reference would be helpful. That being said, it seems rather unlikely that any of this information is necessary to understand what's going on so why include it? (en)
- what are "terminal repeats"? (en)
- Is this article about transposon sequencing or Tn-seq? If it is about Tn-seq, the section should be called "advantages" because it doesn't really list any disadvantages, as far as I can tell (en)
- This refers to bacteria which have been mutated by inserting transposons into their genome? (en)
- "It" = "what"? MmeI? endonuclease? I thought the endonuclease was doing something with the terminal repeat of the transposon? Now the endonuclease is part of the transposon, or part of the terminal repeat of the transposon? So it is interacting with itself? (en)
- Wasn't this already discussed in multiple sections above? Can we choose a single explanation of this and rely on it throughout the rest of the article please? Or is there some additional subtlety introduced in this section which I'm not aware of? (en)
- Didn't the introduction paragraph say that TraDIS is a type of transposon sequencing? Is this article about Tn-Seq or transposon sequencing? Again are Tn-seq and transposon sequencing intended to be understood as synonymous, or is Tn-seq just one choice among a number of protocols implementing transposon sequencing? (en)
- "Library" is a super-jargon-y term in this context which again refers to another analogy which the layperson should not be expected to be familiar with. I have heard members of my lab use it for months now and I still don't understand what they're referring to. (en)
- Isn't this supposed to be the whole point of the method and something that paragraphs were spent trying to describe above? Why is this only allocated a relic sentence at the end of the paragraph? How does it relate to any of the biochemistry mentioned above? I.e. how does any of the above allow us to look at the effects of the insertion of gene function? (en)
- what does it mean for a transposon to be "stable"? are there "unstable" transposons? what property causes their stability or lack thereof? the sequence? the bacterium's defenses? (en)
- Is the article about "transposon sequencing" or about "Tn-seq"? Are those two terms even synonymous? Is this section talking about advantages of Tn-seq over other protocols for achieving "transposon sequencing" or is it about the advantages or disadvantages of "transposon sequencing" for solving/identifying certain problems compared to other methods? (en)
- Again, is the article about transposon sequencing or about Tn-seq? Are they the same thing? It sounds like this was written by some author of a Tn-seq paper, and sounds really biased and not objective (en)
- are we supposed to understand "transposon sequencing" and "Tn-seq" as being two different things? does one refer to a specific protocol for implementing the other? (en)
- Isn't all PCR amplified DNA isolated using agarose gel? This is confusing because generalities of PCR are being discussed as if they were specific to this method. Thus even for readers familiar with PCR who would understand this better, it's still confusing. Explicitly identify what is unique to this method and which parts are subroutines that are commonly done things. Otherwise the definition/meaning of the method is a formless blob. (en)
- I have some vague idea of what a "primer" is, and what it means for a "primer" to be "specific to X" means, but the average reader probably does not. Anyway does it really matter that a primer was used to amplify the sequence via PCR, since you know basically all PCR amplifications are performed using sequence-specific primers? What is exactly gained in understanding by adding this extraneous information? At the very least link to a Wikipedia article defining "prmer" and PCR (en)
- Again, what is a recognition site, and who is doing the recognizing? IS it the recognition site of MmeI? If it is, why is it called the "recognition site" if MmeI is actually recognizing something 20 base pairs away? Shouldn't that be called the "recognition site" instead? If so, why do we have to talk about this other sequence location? (en)
- What does it mean for base pairs to "flank"? What are they flanking in the first place? And why do we care about these flanking sequences? Shouldn't we care about the transposons or the genes or something? (en)
- Didn't the introduction paragraph say that HITS and TraDIS are types of transposon sequencing? Is this article about Tn-Seq or transposon sequencing? Again are Tn-seq and transposon sequencing intended to be understood as synonymous, or is Tn-seq just one choice among a number of protocols implementing transposon sequencing? (en)
- what on earth is an endonuclease, much less a restriction nuclease, much less a type IIS restriction endonuclease? does it matter that the endonuclease is a restriction endonuclease? does it matter that the restriction endonuclease is a type IIS endonclease? why mention all of this? (en)
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- Transposon insertion sequencing (Tn-seq) combines transposon insertional mutagenesis with massively parallel sequencing (MPS) of the transposon insertion sites to identify genes contributing to a function of interest in bacteria. The method was originally established by concurrent work in four laboratories under the acronyms HITS, INSeq, TraDIS, and Tn-Seq. Numerous variations have been subsequently developed and applied to diverse biological systems. Collectively, the methods are often termed Tn-Seq as they all involve monitoring the fitness of transposon insertion mutants via DNA sequencing approaches. (en)
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- Transposon sequencing (en)
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