Talk:Sequencing

Sequencer rate question

I'm a little bit concerned about this:

Current automated DNA sequencing instruments are able to sequence as many as 384 samples in a batch (run) and perform as many as 24 runs a day.

Is that really true? The most recent sequencer I could find at Applied Biosystems is the 3730xl, which has 96 capillaries. I modified the sentence a while ago to say '384 samples in a batch' since the ABI3700 I have access to can run 4x96 well plates serially, without intervention. I suspect it really should say 96 samples in a batch/block/at once.

Also I think the article should mention that 'sequencing machines' only do the electrophoresis run, the reaction is done in a separate thermal cycler. Unless there are combined machines I don't know about. Loris 17:23, 27 July 2005 (UTC)

I also can not find a sequencer (I only checked ABI) more than 96 capillaries. And I also have never heard of a reaction & electrophoresis coupled machine. So I support any changes you want to make. Adenosine | Talk 22:39, July 27, 2005 (UTC)

Although there are only 96 capillaries, it can handle 384-well plates. The 3730xl is not like the 3700, it has a plate stacker and you could load it with many plates so it could run over the weekend. It can do about 40 runs for the short runs, so ~40*96 samples per day. : 07:04, 13 September 2006 (UTC)

I wrote the first sentence so I will disagree with you both. The MegaBACE 4000 (*http://www4.amershambiosciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&parentid=328332&moduleid=166540) runs 384 samples at once- there are other sequencers out there other than by ABI.

I don't think there are more than a handful of 384-cap Megabase sequencers in the field--at least in the US. I don't know of a single facility that has one. But I do know of hundreds of 3730XL's. Even the previously megabase dominated shop, the JGI, switched to 3730XL's. --Phillip SanMiguel 04:08, 8 November 2005 (UTC)--128.210.192.206 04:00, 8 November 2005 (UTC)

What does the number of MegaBACE 4000 have to do with anything. They exist so the original sentence is correct.

So how do you do the sequencing then?

There's something odd going on here: This article lists Shotgun sequencing and Chromosome walking as methods used to sequence DNA, but they then include statements like "...and then these clones are sequenced" and "2. The new short DNA strand is sequenced", and link back here as though the actual sequencing is done by some other method, and these are - well, something other than sequencing, clearly: meta-sequencing, perhaps? I can only think that what is meant is "...shorter strands are then sequenced using sequencing method X, which only works for short strands", but at the moment they seem to imply that one of the steps of "sequencing" is "sequencing". The only linked method which seems to explain how the sequencing itself occurs is Chain termination method - could this be sequencing method X? Anyone care to clarify? - IMSoP 00:19, 22 Oct 2004 (UTC)

You're right: it's the Chain termination method. Because this method only works for short sequences of DNA (about 100 basepairs), other methods are needed to create short pieces and puzzle them back together. Eef (A) 21:00, 28 Oct 2004 (UTC)

OK, I'll go fix the articles to reflect this. - IMSoP 21:10, 14 Nov 2004 (UTC)

ok, I did fix it. I think now it's clear that chain termination is highlighted as the only used technique these days, i know of one other technique that is not used anymore. Confusion about shotgunning being comparable to chain termination is over, i think, the are not in the dame category they utilize each other. Also the first line was redundant, when is sequencing in genetics not restricted to determining the sequence of nucleotides? Sequenceing is deducing the the primary structure of a biopolymer, why added vague confusing language too it. Any ideas on this? -Adenosine- 09:09, Mar 9, 2005 (UTC)

I assume you're talking about Maxam and Gilbert's chemical modification method? I made a note that it is the other DNA sequencing method that was once used. --Kinglz 05:12, 8 Jun 2005 (UTC)

Anonymous comment

The following was moved from the end of the "nucleotide sequencing" section of the article, left there by an anonymous visitor:

(The problem is, this article doesn't really explain *what it is* to a layman. Less technical terms, and a bit of "why do they do this" would be nice...)

Are all dates correct?

asfaik the method Sanger used was developed in 1975

Sanger&Coulson J Mol Biol. 1975 May 25;94(3):441-8. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase.

making the Maxam&Gilbert method not older. (I made a reference here to an article from 1973, but that concerned RNA sequencing). --dol3st 08:00, 14 October 2005 (UTC)

After a chat mith my boss, I think I understand why the Maxam-Gilbert method was used historically, then discarded in favour of the Sanger method, when the two methods were developed at around the same time. Sanger sequencing required cloning of the fragment to be sequenced into a vector to produce single-stranded DNA. This would also have to be done for every subsequent walking step, which may be every 2-300bp or so. Maxam-Gilbert sequencing, on the other hand, could be performed directly on purified DNA. Also, the Sanger method was initially approximately as unwieldy in method - 4 gel lanes, radioactive primer etc. There may also have been some 'politics' involved in the initial takeup.

I've changed the Maxam-Gilbert section to mention this (slightly more briefly). But I took out a date, (1977) because it wasn't clear which method it referred to, and didn't add much anyway.Loris 13:53, 14 October 2005 (UTC)

In agreement with the anonymous commentator

The content of this article is too technical; the basic idea of Sanger sequencing can be communicated very well to anyone with high school biology (indeed, if they didn't learn it in high school biology), and this encyclopedia article should be as accessible as possible. As it stands we seem to assume that the reader cares about di-deoxynucleotides and immediately understands "size-separated by electrophoresis in a slab polyacrylamide gel" without further explanation.

I think the technical/procedural details should be dropped in favor of a more cartoonish explanation of how it works. --Mike Lin 14:30, 14 October 2005 (UTC)

Are you referring to 'anonymous comment' above? If so why not link this thread up so it makes sense?

In any case, while I agree with the intent, I disagree with your suggestion. Indeed, it seems to be contrary to the direction your own edits are taking it (for example changing "DNA sequencing can only be used to determine relatively small regions of a particular DNA template at a time" to "Because of the geometrically decreasing probability of chain termination at increasing lengths, as well as physical limitations on the size and resolution of the gel, the Sanger method can only be used to sequence relatively small fragments of DNA at a time")

I think that the ideal article should be useful to everyone, and it would be foolish to delete all that useful content - most of which is I'd say at about undergraduate level, and hence probably quite important detail for a large subset of the likely audience.

If you think a less technical, quick explanation is a good idea (and I agree that it would be), then perhaps the initial section (Overview) could be expanded to give this.Loris 23:19, 15 October 2005 (UTC)

My rewrite of the sequencing strategies section is considerably easier to understand than what preceded it. I propose to move the technical details about Sanger sequencing in this article into Chain termination method and leave an overview comparable to the other sections. This will make the article more balanced overall. Even still, an easier to understand overview is needed in Chain termination method. --Mike Lin 00:55, 16 October 2005 (UTC)

I disagree on several points. Firstly that your rewrite of Sequencing strategies was easier to understand - but lets get over that, because we are both biased, but more importantly it has been iteratively modified by both of us and is I think better now than it was before.

Secondly, your suggested merge. With respect to your suggested redirect to Chain termination method, The article there is (in my opinion) quite inferior to what is present in the "Sequencing" article. So any merge should replace what is present there, for the most part.
Also, there is a lot more to the article than just current, automated Sanger sequencing – it goes over the history of it, other methods, the current state of the art and so on. I think there is a case to have all this stuff together. Look, for example in the other sequencing-related discussion pages for the confusion about the terminology of sequencing; sequencing reads were confused with the strategies. This could be resolved by making this the overarching article, referring to and being referred by the others. Many of these other points are too short to have articles in their own right, and gutting out the current method could leave a set of links, followed by the stuff which didn’t fit in anywhere else. Which doesn’t really make for a satisfactory article.

Is it that you basically object to an ‘imbalance’ between DNA and everything else? I'd prefer to see the protein sequencing section of this article expanded slightly, but I am myself not qualified (or inclined) to do it.

Also, I think that title "Chain termination method" is rather imprecise. "Chain termination sequencing" would be better. Would you consider renaming it and 'merging' to that instead? I do agree that the precise detail could go to another page, but if you want to move stuff out of this article, I think you(/we) should get the replacement done first! A ‘simpler’ explanation for the layman would almost certainly require a set of new diagrams. Also I think it would need to be clear that the articles had different targets, because otherwise future changes would lead to the same problem recurring a few dozen minor changes down the road. Given all this, my preference would be to just have the simple explanation at the top, followed by the detailed reference below, possibly with the few good non-redundant bits of chain termination method merged in.Loris 17:14, 17 October 2005 (UTC)

DNA sequencing and protein sequencing should be based in separate articles because they are separate subjects. People sequencing DNA and people sequencing protein are using completely different techniques and equipment, and are often trying to solve different fundamental problems (genomics vs. proteomics). It's god to have a broad topic article (e.g. Genetics) to tie these together, but they're basically separate subjects. With regards to Chain termination method, I think there is worthwhile content in there (none of which I wrote), and the merge would be a merge. No objection to renaming it or even turning the DNA sequencing redirect into an article so as to (as you suggest) be inclusive of the history and other approaches. --Mike Lin 02:31, 18 October 2005 (UTC)

Well, I don't think protein and DNA sequencing is so fundamentally different. Certainly they use different procedures, but the reason for the 'sequence' article in the first place is because they are theoretically the same. OK, so protein sequencing is sometimes done on a sample to determine if it has been processed, but I believe it is common to end-sequence to determine just what it is you have, then go to the DNA sequence to find out more (screening and generating it if necessary).

Nevertheless, I don't have a problem with such a cleavage plane. There is some good content in "Chain termination method", but some is confusing or inaccurate. In particular, it doesn't manage a good description of how the technique evolved, but instead mixes them up together in a contradictory mess. So I suggest you take from that article with caution. The "sequencing" article could do with a start-to-finish going over also, so the merge could be quite time consuming. Also, you originally wanted a cartoon-like description of sequencing, and that would presumably go in with the 'DNA sequencing' article as a section rather than in generic sequencing (which would presumably become a list of very brief descriptions and links). The 'why sequence' section would be left behind, or replaced by individual sub-sections, perhaps.Loris 13:38, 18 October 2005 (UTC)

Capillary Sequecing explanation

Either the section on dye-termination or automated sequencing should include the basics of how the current capillary-based sequencers function. That is: dye labeled fragments are drawn into a capillary tube by electrophoretic current, as these fragments pass a "window" in the capillary a laser is fireds through which causes the labeled bases to emit light at a different wavelength which is detected by a "camera" and sent to a computer where the data is computed into a chromatogram with all of the relative intensities of each base.

Contradiction in the article

Under DNA sequencing it says: "More genome data is now being produced by pyrosequencing than Sanger DNA sequencing." Still, under Sanger sequencing it says "This method is now used for the vast majority of sequencing reactions as it is both simpler and cheaper. " At least one of these statements need to go. I know both methods are widely used so they may both be too bold. Akita86 (talk) 11:16, 19 January 2009 (UTC)

Title of the article

It is best to make the name of the article more specific and to change it in "DNA sequencing". There are other fields of technology where the concept of "sequencing" is also used. 193.191.180.232 (talk) 09:05, 20 July 2009 (UTC)

The article "DNA sequencing" already exists. This article appears to essentially be a hybrid of pages on DNA, RNA and protein (and polysaccharide) sequencing, which is of questionable value. It seems the main value of the page would be in clarifying what "sequencing" means in the context of biology, and leaving a detailed description of methods for the specific pages dedicated to these practises. Maonao (talk) 11:36, 23 July 2017 (UTC)