Metagenomics: we have all the answers (they're just all different)

I'm just on my way back from a JGI workshop on metagenome informatics, and I thought I'd take the opportunity to write up a short review.

The workshop was, frankly, excellent. We saw a bunch of talks on metagenome assembly (my current interest) as well as single-cell sequencing approaches, and a whole spate of data analysis platform talks. The basic perspective seems to be this: nobody has the solution to everything, and those who do have some answers seem to get different answers from others. The one possible exception to this was Jill Banfield, who gave a really inspiring talk about how they're using metagenomics on low- and medium-complexity communities to do some excellent science.

(Advertisement: my talk on scaling metagenome assembly is online here, at slideshare.)

The only somewhat dissatisfying note to the whole conference was the overall lack of inter-project cooperation and cross-validation. While some of the analysis platform folk are talking to each other, I don't think they have a good handle on how to really test each other's software, much less combine forces; too much of it all boils down to "trust us -- our approaches work", which is simply not the way to do science. Period.

Pain

"The thing that is driving all of us is pain." -- Victor Markowitz

This single statement probably sums up the workshop best! One of the most obvious tensions in the workshop was between sensitivity of results and scaling. For many microbial populations, it is critical to sequence deeply in order to see rare variants and species; and yet our assembly and annotation tools are, generally speaking, unprepared to handle this volume of data (dozens of Gb, if not Tb). This leads to pain, as we desperately attempt to make use of the data to address our biology.

There were also quite clearly two camps of people. One group had experience with metagenomic data sets of simple- to middling-complexity: think human microbiome, with up to a few hundred species of microbes per sample. The other group was confronting water and most especially soil, which may have hundreds of thousands if not millions of species per sample. The first group was prone to saying things like "it's not that tough a problem, folks! you just need to analyze more sequence! and then you can do anything you want!" to which the second group would then say "yeah, that's the problem, innit? all that sequence?"

I think in a year or so it will be easier to characterize this gap in complexity. We're finally getting results from soil assembly, and it's clear that we need terabases of sequence to get good results; but we don't yet have the ability to quickly & confidently analyze those terabases of sequence. Once we do, we can make quantitative statements illustrating the divide.

It was also nice to hear that everyone had settled on the best possible data processing pipeline at the meeting! (Although perhaps coincidentally, it was almost always their own software.)

Standards

The second day featured a lot of discussion of standards. Many people seemed to have a community standard that, if everyone would just start using theirs, would solve all the problems!!!

Of particular interest to me, Owen White gave a talk where he presented on the Open Source Data Framework. This is basically a central-server NoSQL implementation for storing metadata and data together. Eventually it will support data migration for locality, and lots of other nice features. The response to building Yet Another Big Database (But This Time, With No Schema!!) was muted, although I'm personally quite bullish on the idea that maybe, just maybe, we can have a place where the data and metadata are combined (!!!). It seems that the alternative to an OSDF-like database is to continue using flat files -- maybe even with URLs sometimes!! -- and I see that approach continuing to engender chaos. An alternative would be nice.

(I hate SQL for dataset storage, but maybe that's not a majority opinion.)

One particularly good quote from Victor Markowitz on the OSDF proposal, paraphrased: "This system will let us spread misunderstandings further and more quickly!" Hmm, is that a negative?

Also, someone said the HMP has 14 trillion reads, or 1.4 petabases of sequence. Whuh? Apparently this is what IMG/HMP and METAREP are being built to analyze. Yikes.

Big Black Data Analysis Boxes

Big black boxes for data analysis is a running theme in metagenomics, although I'm not entirely sure why -- something in the water? (We should sequence that.) Things like MG-RAST, IMG/M, CAMRA, and the various K-base projects offer to take your data (raw or not); fold, spindle, and mutilate it; and then return Results.

This approach gives me the heebiejeebies. I have lots of questions: What software are they running, how, with what parameters? What kind of internal QC do they have, and how can I run it myself on my own data? What version of what databases are used? What filtering do they do? The response (more or less) always seems to be "TRUST us! We know what we're doing!"

That line works better in a sitcom than it does in real life.

I really, really want to see a platform mentality evolve. Cue Steve Yegge's "platform" rant: http://news.ycombinator.com/item?id=3101876 or http://steverant.pen.io/

What's the goal, anyway? Reads vs. assembly

Several people were particularly grumpy about this whole "assembly" thing. I can only imagine this stems from having to go to too many meetings where a lot of really theoretical assembler discussion goes on, and is nonetheless treated like the central activity necessary for metagenomics. Since I work on assembly now, I enjoy those talks, but I fully understand that I am a masochist when it comes to science, and not everyone likes assembly.

Nonetheless I think people skeptical of assembly are largely wrong, at least in the overall concept. Assembly is a really important approach in metagenomics for several reasons, which Kostas Mavrommmmmmmmatis laid out the second day: scientifically speaking, assembly gives way better statistical signals for both gene finding and analysis of variation; you can also get linkage information from assembly, which is important for operon analysis. And, just as important -- from a pragmatic perspective, the reduction in data size from assembly saves both space and computation time, and assembly smooths out errors in way that not much else does.

That's not to say assembly is easy, or a panacea, but I think it's an incredibly valuable approach for all those reasons.

As for single read analysis, I am deeply skeptical of any conclusions reached from analyzing reads of length 120 or less -- that's not much signal, bub. (Note, I'm also guilty of this; check out my publication with Victoria Orphan in 2006. What can I say? I was younger and naiver.) Friends don't let friends analyze Illumina short reads. And yet, it seems to be a prevalent, perhaps even dominant, activity in metagenomics. Boo.

So: assemble, man. It's needed.

But as for goals... well, Brooklin Gore said it well when he said, "You come to understand that the purpose of a machine is ultimately not to run programs, but to solve problems." What problems do we want to solve?

Good question.

There wasn't much unity in the goals of the attendees. Some people wanted whole genomes. Others wanted genes. Yet others wanted variations in those genes. Presumably most people wanted metabolic profiles of one sort or another, except for those who didn't. I think this reflects something that Guy Cochrane pointed out: sequencing is fast becoming a common feature to much of biology, and it is extremely hard to address everyone's needs in one platform. So, that makes it even more unlikely that one single approach or one single analysis platform will answer even a majority of users's needs. There's a lot of room out there, folks.

On benchmarking

Everyone got up and gave a different set of assembly results, and it became kind of obvious that everyone optimizes their assembler for a specific set of statistics and then reports only those. Hey -- what about a standard set of benchmarks??

Well, there was a strange reluctance on the part of some Senior Scientists to invest in assembly benchmarking. As near as I could make out, this was because of a fear that assembler authors would then dive into optimizing for those benchmarks at the expense of Platonic truth. Fair 'nuff. But I think there's gotta be a happy medium between No Benchmarks and Only Benchmarks. Right now I find it extremely difficult to figure out which assemblers are better for what purposes, and I'm pretty sure everyone else is just as confused (modulo the authors of assemblers themselves, who are generally quite positive that their assembler is the best).

Anyhoo, I'm thinking quite hard about setting up a couple of single cell and metagenomic data sets, along with assembly and evaluation pipelines, on Amazon Web Services. That should at least make it easy to run people's assemblers on the same data sets & compare the results.

--

Hmm, conclusion time...

The metagenomic and single cell data is already coming, thanks to our sequencing center overlords.

Generally speaking, we don't know how to handle it, either by assembly or by single read analysis. We certainly don't know how to scale all the analyses that everybody wants to do.

The data will still be coming, regardless.

Good times!

--titus

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