Gas station without pumps

2011 May 31

A use for an Ion Torrent

Filed under: Uncategorized — gasstationwithoutpumps @ 09:21
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I’ve been wondering what an Ion Torrent sequencer is useful for.  I mainly deal with de novo assembly of genomes, which needs a lot more data than an Ion Torrent sequencer provides, even for assembling bacterial genomes.  The high error rate and relatively short read length of the Ion Torrent reads is also a problem.  For de-novo sequencing, almost everyone is going with the Illumina platform, which provides barely long enough reads (a little over 100-long at each end of a pair) at the lowest cost.  I like to have some longer 454 reads to throw into the mix, but they are more expensive and confuse some of the de Bruijn graph assemblers.

This past week, though, I’ve been working on a problem that might be ideal for the Ion Torrent: assembling the mitochondrial sequence of the banana slug, Ariolimax dolichophallus.  I’ve been trying to assemble it from 10x whole-genome shotgun sequence using Illumina reads (with paired ends that were too close together, so many of the reads overlap in the middle).  The library prep looks like it was very good at excluding mitochondria:  the mitochondrial genomes seem to have little more coverage than the nuclear genome. [Correction: I must have dropped a decimal point somewhere—the coverage is indeed much higher for the mitochondrion: more like 200x than 10x.]

Since mitochondrial genomes are the primary way of identifying eukaryotic species (often using only a tiny snippet, the “barcode of life“), there is a lot of value in being able to determine the genome quickly and cheaply. A mitochondrial genome is much shorter than bacterial genomes (only about 15 kbases), which makes the low coverage, short reads, and high error rates not much oof a problem. If you have over 100x coverage on a short genome, you can still align and assemble it despite the noise, especially since repeats are not a problem in mitochondria.

Isolating mitochondrial DNA is also supposed to be relatively easy, so it might be good for Ion Torrent to put out a “mitochondrial genome kit” that makes isolating the mitochondrial DNA, sequencing it, and assembling the resulting genome very cheap.  This would take the rather thin taxonomic sampling of 2654 mitochondrial genomes at NCBI to hundreds of thousands in just a few years.  The key thing is to make the library prep very cheap and simple, since otherwise one could do barcoding to multiplex samples and piggyback on sequencing runs on the larger batch machines.

Average error rate and length of Ion Torrent reads after trimming off the bad bases at the end of the read. Note: this is not my data, so I can't provide any information about the library prep, regents or chips used, or any of the other information that may have a major bearing on the quality of the data.  Anyone who has Ion Torrent data that has a reference genome that can be mapped to can do a similar plot.  It would be useful to do such plots for all the platforms in common use, but I don't have the data for it.

For this data, the error rate on 50-base-pair reads is about 1%.  That is much worse than Illumina (which gets 1% around 90 base-pair reads) or 454 (which gets 1% around 400 base-pair reads).  Note that the Q values are reasonably (perhaps slightly optimistically) calibrated in that an error rate of x needs a cutoff about -10 log_10 x

I expect that Ion Torrent will improve their read length and accuracy, but without plots like this one, it is difficult to compare how platforms really perform.  The raw "number of bases" and "read length ignoring error" figures that get touted by the companies are so misleading as to verge on fraud.

 

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9 Comments »

  1. Can you provide some more details about the data that were used to generate this plot, and how the trimming was carried out? I realise you included the disclaimer about how library prep etc. were carried out, but it would be interesting to find out if, for example, this were from the E. Coli installation run, or from PCR products. It is hard to assess what this really means without a little more information. Even with out this additional information, thanks for posting the plot. The paucity of publicly available experimental data makes it hard to draw real conclusions about the claims being made for the Ion platform.

    Comment by Andy May — 2011 May 31 @ 13:47 | Reply

    • The DNA was genomic bacterial DNA from an organism we had sequenced with 454 reads. My contribution to this project was the assembly of the reference genome from the 454 data, and a little help in coming up with useful ways to present the Ion Torrent data.

      Error rates were determined from mapping to the assembly of the 454 data. I did not do the mapping, but a couple of different mappers were used (with slightly different results). I don’t remember which mapper produced this curve. Since Ion Torrent blocked the publication of the paper with the threat of a lawsuit, I stopped working with any data from Ion Torrent machines. I have no use for data that can’t be published.

      Trimming was crude end trimming, chopping the rest of the read off as soon as the Q value dropped below threshold. I did not do the trimming either, so I don’t know whether a standard tool or a custom script was used.

      Comment by gasstationwithoutpumps — 2011 May 31 @ 14:07 | Reply

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  3. Incidentally, I’ve heard through the grapevine that my suggestion for a mitochondrion kit for the Ion Torrent is being considered by the engineers there.

    Comment by gasstationwithoutpumps — 2011 July 4 @ 16:38 | Reply

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