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Using the Illumina platform, it is cheap and (relatively) easy to sequence huge amounts of DNA or RNA. There are various other platforms out there (Roche/454, SOLiD, PacBio, Ion Torrent) each with their own distinct advantages, but Illumina seems to be pretty popular for many applications, despite its limitations.

Ideally, we would like a sequencing technology that produces long, error-free reads with high throughput. However, at this point it seems we have to make a choice: throughput or length (and quality). PacBio seems promising, but the last I heard they have still been unable to deliver on their claims.

What are the molecular and biochemical limitations to our current sequencing technologies? Why don't we already have long, error-free reads with high throughput?

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I suspect the project triangle is at work here. en.wikipedia.org/wiki/Project_triangle –  kmm Feb 1 '12 at 3:06
    
@bobthejoe Ideally, we would like as long as possible, right? :) I consider the upper limit of Sanger read lengths (+/- 1000 bp) to be long, but it would also be nice to know why we can't get longer reads (with or without high throughput, low error rates, so on). –  Daniel Standage Feb 3 '12 at 21:15
    
All or most of the limitations you describe can be simply overcome by brute force method. Almost any genome sequenced on any high throughput technology today will be of very high quality if you sequence it 100 times. It's just a matter of time and money. Remember the original human genome was sequenced by many Sanger machines and a lot of cloning. –  yotiao Apr 18 '12 at 12:54
    
@yotiao What you're saying is true, but only to a point. My experience is that assemblies and other analysis improve with coverage, but at a certain point adding more data provides only minimal improvements, even if there is a significant addition of new data. And to say that the human genome project was successful primarily because they dedicated a lot of Sanger sequencers to the project ignores the significant differences between the nature and quality of the data produced by the Sanger platform as opposed to today's high-throughput platforms. –  Daniel Standage Apr 18 '12 at 13:07
    
@Daniel Yes, you're right in principle. But then if what you require is as-perfect and complete a sequence as possible, then I'd propose that the limitations are the same as they were before: DNA itself, with it's super high repetitive content, poliploidy. Sanger and positional cloning did not manage to solve this problem (hence gaps in finished human genome sequence) and they're regarded as the golden standard (am I that old already?). –  yotiao Apr 18 '12 at 13:20
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2 Answers 2

It seems like you answered your own question,the signal from a few molecules running through an enzyme or a polymerase tend to fall out of synch after a few hundred bases. If an enzyme for sequencing was more rigorously in time step that could help for instance. The machines read traces in four channels with nice bumps for each base. See this article for a nice example. You can see that if there are too many of the same base consecutively it gets hard to tell how many bases there are there. Over time all four traces will start to smear out and you cant tell Adam from Thelma if you take my meaning.

But there are other bottlenecks.

The sequencers currently put out such a high volume of data that the analysis of the meaning of the output data can't be analyzed fast enough. This follows the trend in biotech over the past 12 years or so - more sequence data, micro array data, more mutation data, more genomes than people who can actually use it to understand the biology. There is a bit of an analysis bottleneck now.

So some of these sequencers have greater read lengths, which can make it easier to assemble a sequence. These sequencers generally cost more. For instance if you have a library to sequence a little fungal or algal genome - you will get the answer back in a day or less now. In the form of 1 Tb of reads maybe 50 to 200 bp long. It might take quite a bit of time to put that together into a novel genome sequence, still more to find the genes, build the gene networks from a template of pathways etc. Just imagine a thousand sequencers pumping out day and night and you get the picture I'm trying to paint here.

About cost. Ion Torrent and the new oxford nanopore sequencers are really cheap - \$50k to perhaps \$900 for Oxford Nanopore's USB sequencer. Most other systems cost hundreds of thousands of dollars. Ion torrent and Nanopore have more disposables - you throw away a chip or even the entire sequencer - at a cost of hundreds of dollars a sample.

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Over the summer I've been to a couple of conferences including a day seminar focused on assembly of 30x coverage genomes from MiSeq data and the biases in the sequencers were creating systematic errors that made some of the data difficult to interpret. HTS data is not plug and play, even with open source software. not yet. –  shigeta Sep 5 '12 at 14:38
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Q1) What are the molecular and biochemical limitations to our current sequencing technologies?

A1) AFAIK:

Illumina has hard time producing long reads (although now miseq can generate reads that are 300bp and that can be paired, the so called paired end 2X300) because after a certain number of bases that are synthesized and recorded on camera (Illumina is sequencing by synthesis, basically you add bases and measure fluorescence at each cycle), i.e. after a certain number of "cycles" you can lose syncro, and the quality of bases decreases.

PacBio can generate very long molecules, but they still have big problems with reliability of the reading of the bases (I don't know what's the problem here)

Q2) Why don't we already have long, error-free reads with high throughput?

A2) Because it's hard to do! But we are moving towards this!

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