Microbial community structure and dynamics in thermophilic composting viewed through metagenomics and metatranscriptomics

Composting is a promising source of new organisms and thermostable enzymes that may be helpful in environmental management and industrial processes. Here we present results of metagenomicand metatranscriptomic-based analyses of a large composting operation in the Sao Paulo Zoo Park. This composting exhibits a sustained thermophilic profile (50 degrees C to 75 degrees C), which seems to preclude fungal activity. The main novelty of our study is the combination of time-series sampling with shotgun DNA, 16S rRNA gene amplicon, and metatranscriptome high-throughput sequencing, enabling an unprecedented detailed view of microbial community structure, dynamics, and function in this ecosystem. The time-series data showed that the turning procedure has a strong impact on the compost microbiota, restoring to a certain extent the population profile seen at the beginning of the processand that lignocellulosic biomass deconstruction occurs synergistically and sequentially, with hemicellulose being degraded preferentially to cellulose and lignin. Moreover, our sequencing data allowed near-complete genome reconstruction of five bacterial species previously found in biomass-degrading environments and of a novel biodegrading bacterial species, likely a new genus in the order Bacillales. The data and analyses provided are a rich source for additional investigations of thermophilic composting microbiology.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Provost's Office for Research of the University of Sao PauloCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Sao Paulo, Inst Quim, Dept Bioquim, Sao Paulo, BrazilUniv Sao Paulo, Programa Pos Graduacao Interunidades Bioinformat, Sao Paulo, BrazilUniv Sao Paulo, Escola Artes Ciencias & Humanidades, Sao Paulo, Brazil|Fundacao Parque Zool Sao Paulo, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Ciencias Biol, Sao Paulo, BrazilBiocomplex Inst Virginia, Blacksburg, VA USADepartamento de Ciências Biológicas, Universidade Federal de São Paulo, São Paulo, BrazilFAPESP: 2011/50870-6Web of Scienc

Multiple alignment of large eukaryotic genomes with highly fragmented assemblies

O advento do sequenciamento de nova geração (NGS - Next Generation Sequencing) nos últimos anos proporcionou um aumento expressivo no número de projetos genômicos. De maneira simplificada, as máquinas sequenciadoras geram como resultado fragmentos de DNA que são utilizados por programas montadores de genoma. Esses programas tentam juntar os fragmentos de DNA de modo a obter a representação completa da sequência genômica (por exemplo um cromossomo) da espécie sendo sequenciada. Em alguns casos o processo de montagem pode ser executado com maior facilidade para organismos com genomas de tamanhos pequenos (por exemplo bactérias com genoma em torno de 5Mpb), através de pipelines que automatizam a maior parte da tarefa. Um cenário mais complicado surge quando a espécie possui genoma com grande comprimento (acima de 1Gpb) e elementos repetidos, como no caso de alguns eucariotos. Nesses casos o resultado da montagem é geralmente composto por milhares de fragmentos (chamados de contigs), uma ordem de magnitude muito superior ao número de cromossomos estimado para um organismo (comumente da ordem de dois dígitos), dando origem a uma montagem altamente fragmentada. Uma atividade comum nesses projetos é a comparação da montagem com a de outro genoma como forma de validação e também para identificação de regiões conservadas entre os organismos. Embora o problema de alinhamento par-a-par de genomas grandes seja bem contornado por abordagens existentes, o alinhamento múltiplo (AM) de genomas grandes em estado fragmentado ainda é uma tarefa de difícil resolução, por demandar alto custo computacional e grande quantidade de tempo. Este trabalho consiste em uma metologia para fazer alinhamento múltiplo de genomas grandes de eucariotos com montagens altamente fragmentadas. Nossa implementação, baseada em alinhamento estrela, se mostrou capaz de fazer AM de grupos de montagens com diversos níveis de fragmentação. O maior deles, um conjunto de 5 genomas de répteis, levou 14 horas de processamento para fornecer um mapa de regiões conservadas entre as espécies. O algoritmo foi implementado em um software que batizamos de FROG (FRagment Overlap multiple Genome alignment), de código aberto e disponível sob licença GPLv3.The advent of Next Generation Sequencing (NGS) in recent years has led to an expressive increase in the number of genomic projects. In a simplified way, sequencing machines generate DNA fragments that are used by genome assembler software. These programs try to merge the DNA fragments to obtain the complete representation of the genomic sequence (for example a chromosome) of the species being sequenced. In some cases the assembling process can be performed more easily for organisms with small-sized genomes (e.g. bacteria with a genome length of approximately 5Mpb) through pipelines that automate most of the task. A trickier scenario arises when the species has a very large genome (above 1Gbp) and complex elements, as in the case of some eukaryotes. In those cases the result of the assembly is usually composed of thousands of fragments (called contigs), an order of magnitude much higher than the number of chromosomes estimated for an organism (usually in the order two digits), giving rise to a highly fragmented assembly. A common activity in these projects is the comparison of the assembly with that of another genome as a form of validation and also to identify common elements between organisms. Although the problem of pairwise alignment of large genomes is well circumvented by existing approaches, multiple alignment of large genomes with highly fragmented assemblies remains a difficult task due to its time and computational requirements. This work consists of a methodology for doing multiple alignment of large eukaryotic genomes with highly fragmented assemblies, a problem that few solutions are able to cope with. Our star alignment-based implementation, was able to accomplish a MSA of groups of assemblies with different levels of fragmentation. The largest of them, a set of 5 reptilian genomes where the B. jararaca assembly (800,000 contigs, N50 of 3.1Kbp) was used as anchor, took 14 hours of execution time to provide a map of conserved regions among the participating species. The algorithm was implemented in a software named FROG (FRagment Overlap multiple Genome alignment), available under the General Public License v3 (GPLv3) terms