A few weeks back, Nick Loman (via Manoj Samanta) brought MEGAHIT to our attention on Twitter. MEGAHIT promised "an ultra-fast single-node solution for large and complex metagenome assembly" and they provided a preprint and some open source software. This is a topic near and dear to my heart (see Pell et al., 2012 and Howe et al., 2014), so I was immediately interested - especially since the paper used our Howe et al. data set to prove out their results. (The twitterati also pointed out that the preprint engaged in some bashing of this previous work, presumably to egg me on. ;)
So I thought, heck! Let's take MEGAHIT out for a spin! So my postdoc Sherine Awad and I tried it out.
tl; dr? MEGAHIT seems pretty awesome to me, although IDBA and SPAdes still seem to beat it by a bit on the actual assembly results.
Installing MEGAHIT
We ran into some small compilation problems but got it working on an Ubuntu 12.04 system easily enough.
Running it was also a snap. It took a few minutes to work through the required options, and voila, we got it running and producing results. (You can see some example command lines here.)
First question --
How does it do on E. coli?
One of the claims made in the paper is that this approach performs well on low-coverage data. To evaluate this, I took a 5m read subset from the E. coli MG1655 dataset (see Chitsaz et al., 2011) and further subsetted it to 1m reads and 500k reads, to get (roughly) 100x, 20x, and 10x data sets. I then ran MEGAHIT with default parameters, specifying 1 GB of memory, and limiting only the upper k size used (because otherwise it crashed) -- again, see the Makefile.
For comparison, I also ran SPAdes on the lowest-coverage data, looking only at the contigs (not the scaffolds).
After it was all done assembling, I ran QUAST on the results.
| Measure | 100x | 20x | 10x | 10x (SPAdes) |
|---|---|---|---|---|
| N50 | 73736 | 52352 | 9067 | 18124 |
| Largest alignment | 221kb | 177kb | 31kb | 62kb |
| bp in contigs > 1kb | 4.5mb | 4.5mb | 4.5mb | 4.5mb |
| Genome fraction | 98.0% | 98.0% | 97.4% | 97.9% |
| Misassembled length | 2kb | 40.8kb | 81.3kb | 63.6kb |
(Data: MEGAHIT 100x, 20x, and 10x; and SPAdes 10x.)
In summary, it does pretty well - with even pretty low coverage, you're getting 97.4% of the genome in contigs > 500bp (QUAST's default cutoff). Misassemblies grow significantly at low coverage, but you're still only at 2% in misassembled contigs.
In comparison to SPAdes at low coverage, the results are ok also. SPAdes performs better in every category, which I would expect -- it's a great assembler! - but MEGAHIT performs well enough to be usable. MEGAHIT is also much, much faster - seconds vs minutes.
Next question -
How well does it do on on a metagenomic data set?
Sherine has been running benchmarks for Velvet, SPAdes, and IDBA on the data set from Shakya et al, 2013, a mock community data set. So I asked her to add some MEGAHIT results. She did quality trimming as specified in Kalamazoo, and ran MEGAHIT with 10 GB of RAM. She then used QUAST to evaluate the results against the known good genomes.
| Measure | MEGAHIT | SPAdes | IDBA |
|---|---|---|---|
| # contigs > 1kb | 19,982 | 16,387 | 16,191 |
| length in contigs >1kb | 190.7mb | 192.5mb | 194.8 |
| # misassemblies | 698 | 894 | 1223 |
| Bp in misassemblies | 12.7mb | 28.2mb | 21.8mb |
| Metagenome fraction | 89.96% | 90.42% | 90.97% |
Again, the answer is "MEGAHIT works pretty well." Fewer misassemblies, but also more contigs and a bit less coverage of the known genome.
Third question --
How fast and memory efficient was MEGAHIT?
Very. We didn't actually measure it, but, like, really fast. And low memory, also. We're doing systematic benchmarking on this front for our own paper, and we'll provide details as we get them.
(We didn't measure MEGAHIT's performance because we don't have numbers for SPAdes and IDBA yet. We didn't measure SPAdes and IDBA yet because actually doing the benchmarking well is really painful - they take a long time to run. 'nuff said :)
So, what are your conclusions?
So far, +1. Highly recommended to people who are good at command line stuff and general all-around UNIX-y folk. I'd want to play around with it a bit more before strongly recommending it to anyone who wasn't a seasoned bioinformatician. It's rough around the edges, and I haven't looked at the code much yet. It also breaks in various edge cases, but at least it's pretty robust when you just hand it a straight up FASTQ file!
That having been said, it works shockingly well and is quite fast and memory efficient. If you're having trouble achieving an assembly any other way I would definitely recommend investing the time to try out MEGAHIT.
--titus
- p.p.s. Thanks to Rayan Chikhi and Lex Nederbragt for reading and commenting on
- a draft version of this post!
Appendix: MEGAHIT and digital normalization
In the MEGAHIT paper, they commented that they believed that digital normalization could lead to loss of information. So I thought I'd compare MEGAHIT on 100x against MEGAHIT and SPAdes running on digitally normalized 100x:
| Measure | 100x | DN (w/MEGAHIT) | DN (w/SPAdes) |
|---|---|---|---|
| N50 | 73736 | 82753 | 132872 |
| Largest alignment | 221kb | 222kb | 224kb |
| bp in contigs > 1kb | 4.5mb | 4.5mb | 4.6mb |
| Genome fraction | 98.0% | 98.1% | 98.2% |
| Misassembled length | 2kb | 120kb | 48kb |
(Data: MEGAHIT 100x, MEGAHIT DN, and SPAdes DN.)
The short version is, I don't see any evidence that diginorm leads to incompleteness, but clearly diginorm leads to lots of misassemblies when used in conjunction with MEGAHIT or SPAdes on high-coverage genomes. (We have some (ok, lots) of evidence that this doesn't happen with lower coverage genomes, or metagenomes.) That having been said, it's clearly rather assembler-specific, since SPAdes does a much better job than MEGAHIT on dn data.
The shorter version? You probably won't need to use diginorm with MEGAHIT, and you shouldn't. That's OK. (There are lots of reasons why you shouldn't use diginorm.)
I still don't have any evidence that diginorm drops information in non-polyploid situations. Let me know if you've seen this happen!
Appendix II: Running your own evaluation
All of the E. coli numbers above are available in the 2014-megahit-evaluation github repo. See README.md in that repo for basic install instructions, and Makefile for what I ran and how to run it. Feel free to reproduce, extend, and update!
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