from Scientific American By Kevin Bonham
…Modern sequencing methods can generate millions or even billions of these short sequence “reads” at a time, but as I said above you need many more than one read per base, and even a single genome often has millions of bases. If you try to read the entire genome of every microbial member of a complex community, you’re going to need several sequencing runs. And though prices have dropped a lot since the days of the human genome project, it’s still far from cheap.
But if you want to know the information in a library, you don’t necessarily have to read every page of every book – just getting a list of the titles is probably sufficient. Sure, some of the books are probably obscure, so knowing the title alone won’t tell you everything, but if you’re interested in comparing, say, the diversity of information in New York Public libraries vs those in Massachusetts, lists of titles in plenty. This is the idea behind 16S ribosomal profiling – essentially going through the pile of fragments that came out of your wood chipper and only looking at the spines.
In the same way that every book has a title, every bacterium has a gene for the RNA component of the 16S Ribosome – a molecule necessary for making proteins. Also useful, this gene doesn’t change very much in bacterial evolution, so the degree of difference between the 16S genes of two microbes is a good proxy for how distantly related they are. And the best part: sequencing a few hundred bases of this gene is plenty to extract the necessary information.
This is the way that a huge number of microbial ecology papers are done – if you see a pie chart with different colors representing different microbes, it was probably done with 16S ribosomal profiling.
Maybe knowing the level of sophistication of the books in NY vs MA libraries isn’t what we’re after, we want to know the level of sophistication of the patrons. Maybe NY libraries have a bunch of Shakespeare and Rumi, but the people going into the library are only reading E.L. James. What we really want to be able to do is analyze what books are being pulled off the shelves.
This is the idea behind RNAseq, which looks at the relative abundance of – you guessed it! – RNA. When genes are turned on in a cell, they make copies of the DNA gene in a molecule of RNA, and the amount of a particular sequence of RNA in a cell is a measure of how much the gene is on. It’s as if patrons of our metaphorical library aren’t allowed to checkout books, they’re only allowed to photocopy the pages of the books they want to read.
Which genes are on or off governs the behavior of a cell more than which genes it has (after all, your heart cells and skin cells have the same genes, but very different behavior), but sequencing the DNA tells us nothing about which genes are actually being expressed, any more than knowing the books in a library tells you about the reading behavior of its patrons. The same technologies I described in part 2 of this series can be turned on the RNA extracted from cells. (read more…)