Better the metagenome you know than the metagenome you don't...

Morgan, J., Darling, A., & Eisen, J. (2010). Metagenomic Sequencing of an In Vitro-Simulated Microbial Community PLoS ONE, 5 (4) DOI: 10.1371/journal.pone.0010209

A new era for the design of metagenomic controls starts! Morgan et al. present the benchmarking of metagenomic tools using artificial "microbial communities" mixed up in the lab.

The Hook...
Metagenomics is a fancy name for what's actually a large and obscure toolbox of molecular biology procedures and computational algorithms that promises to help us in the understanding of whole, natural microbial communities. It is so exciting because it allows us to study organisms (bacteria and archaea specifically) that would otherwise remain unacknowledged because we cannot grow them in the lab. It also provides for the first time the opportunity to analyse whole natural communities, and not only sectors of it (like "granivorous community" or "photosynthetic guild"). The comparison of natural functional communities would help us understand a lot about how communities are assembled, how they evolve and change in time and how are they affected by external disturbances.

Having said that, we still lack the tools to analyse such large databases and the quality standards to produce and compare metagenomes. This happens each time a new technology appears, because there has been not enough time to try and experiment with it as to accurately know its flaws. This is even worse with metagenomics since no whole community has ever been studied and so we don't really know or even suppose how our data should look like. Here's where Morgan et al. come to rescue with a very neat approach.
The Setting...
Their logic is simple and clear: since we do not have any community whose composition is completely known, let's make one. So they retrieved ten different microorganisms from the culture collections whose genomes have already been sequenced, and prepared aliquots so that they would have the same number of cells from each organism. Then they mixed them up, extracted the whole community DNA with three different DNA-extraction protocols and then sequenced four metagenome databases (one was replicated with an alternative sequencing method).

The Bad...
Surprisingly, none of the sequenced metagenomes reflected the original composition of the community mix. This can be caused for a number of reasons: the size of a genome and the number of genome copies per cell affect the probability of sequencing; differences in cell wall and matrix thickness and composition could prevent efficient DNA extraction; specific DNA segments might be harder to clone and/or sequence... When they compared between metagenomes, they found that most differences were due to the type of DNA extraction utilized. That is, the same community will result in different metagenomes when different DNA extraction methods are used. This also means that metagenomes obtained with different DNA extraction protocols should not be compared. Ever.

It still puzzles me one thing: the love for BLAST. Even when they assigned each sequence to a specific organisms by "blasting" each read from the metagenomes to the ten complete genomes of the organisms in the mix, there's a large number of sequences that could not be mapped back to the source organism. Sure, there seems to be a phage infecting some cultures that was not in the sequenced genome. But it is surprising that there was a large number of reads that actually hit a Bacillus, when there were five Lactobacillus strains in the mix. My point is that BLAST is a very poor algorithm to recover precise hits, and the short lenght of the sequences reduce the taxonomic resolution attainable by it, misleading the results. If we add a really biased and incomplete reference database, it ends up being almost impossible to accurately define the genomic composition of a natural community. This also calls for better and more precise methods of assigning or binning of metagenomic sequences.

The Good...
Since "all different" is not a very hopeful result, they prepared three replicas of each DNA extraction method so to say which of them showed a lower variability and hence would be more reliable. It turned out that the DNA kit extraction protocol has a larger repeatability, most likely because there's a lower variation in reagent concentrations.
And then again, although there's large variability inter- and intra- protocol, there are no radical changes in the relative abundance of each organism. That is, there is no change from the dominance of one organism to another. Although they're still not reflecting the "true" abundances.

The Ugly...
One of the samples was sequenced twice, one time with classic Sanger capillary sequencing and the other with pyrosequencing. This helped them to show that differences between extraction methods are far greater than differences between sequencing platforms. Still I sensed a bit of anti-pyrosequencing in it. Sure, pyrosequencing gives shorter reads and so a larger amount of reads will be unassignable to reference organisms (at least by BLAST standards). But I'm not sure that these results actually demonstrate that cloning-bias is not so important. It would be necessary to repeat each sample with pyrosequencing to demonstrate this. And it would be also great to replicate the same example as they did with Sanger. This would actually show how much of this variability is really attributable to DNA extraction and how much of it is attributable to cloning bias.

The Finale...
We desperately need more research like this, that would help us not only to standarize the technology behind metagenomics but also allows to build the robust theoretical framework that metagenomics (and community ecology in general) is so in need. This kind of work should be complemented with in-silico modelations of metagenomes (like that in Mavrommatis et al. 2007), and also with the development of better algorithms to cluster and assign taxonomy to sequenced reads.
After all the metagenomic hype, we still do not know the true structure and composition of sequenced microbial communites. But we do know a lot more than before.

Baños de pureza... y micobacterias

Thanks to Kim Ross from Pace's Lab for pointing this paper out.

Feazel LM, Baumgartner LK, Peterson KL, Frank DN, Harris JK, & Pace NR (2009). Opportunistic pathogens enriched in showerhead biofilms. Proceedings of the National Academy of Sciences of the United States of America, 106 (38), 16393-9 PMID: 19805310

"Cantando en el baño, me acuerdo mucho de tí..." decía Tintán.

Y desde ahora también de Mycobacterium avium (link en español), un grupo de bacterias pertenecientes al grupo de las Actinobacteria. Éstos están caracterizados por tener una compleja pared celular repleta de ácidos micólicos conocida como ácido-resistente [totalmente diferente de las Gram(+) y Gram(-)]. Ésta gruesa pero flexible pared disminuye el tráfico de compuestos entre la célula y su entorno, aumentando la capacidad de retención de agua dentro de la célula y evitando la incorporación de compuestos tóxicos, como por ejemplo antibióticos y antibacteriales, lo que hace de éste organismo una bacteria sumamente resistente.

Pared celular de las mycobacteria

M. avium es lo que se conoce como un patógeno oportunista, es decir que puede infectar humanitos pero sólo cuando éstos se encuentran inmunodeprimidos (con fibrosis cística, VIH, y según algunos doctores, !con depresión!). Tan excéntrico es éste personaje que disfruta de infectar las células fagocíticas (que se comen agentes externos), dejándose "comer" por el fagocito y sobreviviendo con agrado dentro de las vesículas formadas en el interior del fagocito, a pesar del coctél de enzimas vertidas en la misma vesícula con la intención de deshacer al agente infeccioso.

Colonias de Mycobacterium avium

El chiste de ésta entrada es que recientemente anda dando vueltas por toda la media la siguiente noticia: el grupo del Dr. Norman Pace , de la Universidad de Colorado, Boulder, USA (quien también fundó la microbiología ambiental), publicó recientemente en PNAS un artículo en donde reporta un análisis de la diversidad microbiana encontrada en las cabezas de las regaderas de Denver y NewYork, y resulta que el grupo de M. avium y sus parientes es uno de los mejores representados. El problema con M. avium es que generalmente se transmite por aerosoles (es decir, gotitas microscópicas de baba que salen expulsadas al estornudar o toser) que son fácilmente inhalados. Y pues resulta también que la mayoría de las regaderas producen aerosoles al atomizar el agua.

¡Pero que no cunda el pánico!: antes de correr y bañarse con tapabocas o lavar con geles "antiinfluenza" el interior de las regaderas... hay una serie de FAQ's que el laboratorio del Dr. Pace publica en su página, y en resumen dice que no hay que paniquarse, que esto sólo representa un peligro moderado para personas inmunodeprimidas y que simplemente basta con cambiar un par de veces al año la cabecera de la regadera (ojo: las mycobacteriae son generalmente resistentes a los lavados con cloro)... y en caso de que sean súper paranoicos pues tratar de substituír los componentes de plástico por componentes de metal (si claro... unos empaques de hierro son la onda).

En fin... es bonito saber que no sabemos nada y que no tenemos nada sanitizado a pesar de la necesidad de control de ciertas personas que hablan inglés y dominan al mundo.

regadera: dícese de la ducha... para los que no hablan mexica...

y les dejo un video de la noticia en gringolandia:
http://cbs2chicago.com/video/?id=62649@wbbm.dayport.com

Revisión Darwin, Metagenómica y la "Nueva Ecología"

Germán Bonilla-Rosso, Valeria Souza, & Luis Eguiarte (2008). Metagenómica, Genómica y Ecología Molecular: La Nueva Ecología en el Bicentenario de Darwin TIP - Revista Especializada en Ciencias Químico-Biológicas, 11 (1), 41-51

Si de por sí ya uno no podía encontrar ninguna exposición de biólogo en la que no se mencionase a Darwin, pues ahora en el susodicho Año de la Evolución lo vamos a ver hasta en la sopa...

Y yo no voy a ser el biólogo de excepción, así que me autopromociono un poco con éste bonito artículo de colección para la dama y el caballero...

Éste es una breve revisión de metagenómcia y sus recientes avances en metagenómica y la aplicación de las herramientas de la biología molecular a la ecología. A nuestro entender, los enfoques metagenómicos representan una culminación del legado de Darwin como la aproximación al mundo natural visualizando individualmente a los organismos como el producto de su historia evolutiva delineada por su funcionamiento e interacción con su entorno abiótico y con otros organismos.

Y aprovecho para desahogar lo siguiente: hoy día, las publicaciones en español y en revistas no indexadas o de difusión (aunque sea difusión para la misma élite científica) no tienen peso curricular o incluso llegan a valer puntos negativos en el SNI, así que con frecuencia los intentos por producir éste tipo de textos se ven rápidamente desalentados. Éste artículo salió en una publicación de la FES Zaragoza que, hasta donde sé, no se encuentra en la red (por cierto, vale la pena echarle un ojo a esa publicación si tienen oportunidad). Pero ayer recibimos un correo electrónico de una alumna de Coahuila solicitando el texto completo. Curiosamente, mi tesis se desarrolla en Coahuila. Así que me llena de orgullo poner a disponibilidad el pdf nuestro artículo.

¡A escribir en español, pues!

Update: Aquí está el link a la revista, aquí el original.

Metagenome Sequence Simulators

Richter DC, Ott F, Auch AF, Schmid R, & Huson DH (2008). MetaSim: a sequencing simulator for genomics and metagenomics. PloS one, 3 (10) PMID: 18841204


An article from the Huson's Group at Tübingen University has just came out in the Open Access (and scientific publishing innovator) journal PLoS ONE, describing MetaSim, a software to produce artificial or syntetic or in silico metagenomes out of a selection of completely sequenced genomes.

This is just "heaven-sent" for me since I've been working on a set of syntetic metagenomes for the past two months, and will be happy to use this software first hand like... today. It seems that the software not only lets you choose the source genomes from a phylogenetic tree (figures reproduced here from the original article al PLoS ONE thanks to the Creative Commons License), but also choose from three different type of sequencing technology output (Sanger, 454 and Illumina) and generate theorethical metagenome.

This is the continuation of a very important change in genomic sciences, moving from experiments far too expensive or long to be replicated and hence out of hard statistical comparision, to null-model based in silico genomic analysis.

The first effort to analyze the true scope of metagenomic analysis was presented by Kostas Mavrommatis and others from the Genome Biology group at JGI (unfortunately published in an non-OA journal), where they produced three simulated metagenomes of contrasting complexity to asses assembly, gene prediction and annotation (SPOILER: the best combination assesed was Arachne assembler with Fgenesb predictor and PhyloPhytia for binning, and BLAST "performed poorly" as usual). This work also produced a database for the Fidelity of Analysis of Metagenomic Samples (FAMeS), a great effort to standarize metagenomic analysis software. A great alternative is ProxyGene annotation, as reported by the Markowitz group.

Mavromatis K, Ivanova N, Barry K, Shapiro H, Goltsman E, McHardy AC, Rigoutsos I, Salamov A, Korzeniewski F, Land M, Lapidus A, Grigoriev I, Richardson P, Hugenholtz P, & Kyrpides NC (2007). Use of simulated data sets to evaluate the fidelity of metagenomic processing methods. Nature methods, 4 (6), 495-500 PMID: 17468765

I'll play a little with the software and post some of my impressions here... and maybe in the original PLoS ONE webpage since it is totally open to post-publication review!!!!

You can download MetaSim at Huson's Labpage!!!

To Metagenome or not to Metagenome

A masterpiece of science blogging was posted here (http://suicyte.wordpress.com/2007/11/20/smallest-primate-ever-discovered/), addressing the finding of primate sequences in the GOS dataset, just one of the unassesed ambiguities in metagenomics.

The post is beautifully written and made me laugh really loud. The point is that metagenomics is sometimes being overselled just in the very same way as genomics has been (see Eisen's blog for just some examples) and this leads to an skeptic counterwave.

My brief cons and pros:

a) metagenomics offers indeed the unprecedented opportunity to explore unculturable microbial diversity, which no other tool can do.

b) metagenomics is not only a technological advance like genomics was, it fits perfectly in an ecological (community/ecosystemic) theoretical background

c) no matter what everybody says, the GOS sampling has provided an incredible amount of data on previously unknown (and often unimagined) microbial diversity

d) criticisms on the amount of money spent on metagenomics seem to me like questioning the financial support on Humboldt's (or any other naturalist) voyages, which were explorative and not precisely focused on any hypothesis.

e) metagenomics is obviously error-prone, and it's biases have been poorly evaluated

f) metagenomics is much more useful in small, simple communities where a reasonable coverage can be achieved

g) metagenomics is much more useful in well known, deeply studied natural communities where it is employed to answer specific biological questions

h) metagenomics is expensive!

i) a great deal of work is still to be done on: defining parameters for comparing different samples, assessing taxonomical and functional biases, increasing assembly effectiveness and contig construction, improving functional prediction, developing tools for the analysis of such huge datasets, etc.

j) metagenomics is best when interdisciplinary, that is when it's used along with techniques and analyses from other disciplines that might provide physiological, evolutionary or ecological information

That being said, metagenomics rocks!

Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S, Wu D, Eisen JA, Hoffman JM, Remington K, Beeson K, Tran B, Smith H, Baden-Tillson H, Stewart C, Thorpe J, Freeman J, Andrews-Pfannkoch C, Venter JE, Li K, Kravitz S, Heidelberg JF, Utterback T, Rogers YH, Falcón LI, Souza V, Bonilla-Rosso G, Eguiarte LE, Karl DM, Sathyendranath S, Platt T, Bermingham E, Gallardo V, Tamayo-Castillo G, Ferrari MR, Strausberg RL, Nealson K, Friedman R, Frazier M, & Venter JC (2007). The Sorcerer II Global Ocean Sampling expedition: northwest Atlantic through eastern tropical Pacific. PLoS biology, 5 (3) PMID: 17355176

Paper GOS de Venter

Pues tras poco más de un año de estira y afloje, finalmente hoy salió publicada la primera parte de la Global Ocean Sampling Expedition (GOS), en donde el Venter Institute, en colaboración con un buen de instituciones de todo el mundo, explora la diversidad bacteriana marina de la cual se conoce sólo un 1% cultivable. Esta primera parte contiene los datos de Canadá al canal de Panamá, las Galápagos y de ahí hasta la Polynesia Francesa. Una de las muestras es del canal de Yucatán, y está bajo resguardo de la UNAM. Es un evento sin precedentes que según yo equivale a los primeros listados florísticos de Humboldt o algo así. !Y además es mi primer paper publicado! (entre otros dieciocho autores, claro). En fin, vale la pena que todo el mundo le eche un ojo, especialmente si son biólogos o están interesados en los recursos bióticos microbianos.

Felicidades a todos los que se echaron un broncón para que saliera publicado. Especialmente Shibu, Doug y Aaron.

ulk is api

Conferencia de prensa:

Sinopsis de Resultados:

Artículo en PLoS:

Conferencia de Prensa en Vivo HOY: