Analysis of 16S rRNA environmental sequences using MEGAN

Abstract

Background: Metagenomics is a rapidly growing field of research aimed at studying assemblages of uncultured organisms using various sequencing technologies, with the hope of understanding the true diversity of microbes, their functions, cooperation and evolution. There are two main approaches to metagenomics: amplicon sequencing, which involves PCR-targeted sequencing of a specific locus, often 16S rRNA, and random shotgun sequencing. Several tools or packages have been developed for analyzing communities using 16S rRNA sequences. Similarly, a number of tools exist for analyzing randomly sequenced DNA reads.

Results: We describe an extension of the metagenome analysis tool MEGAN, which allows one to analyze 16S sequences. For the analysis all 16S sequences are blasted against the SILVA database. The result output is imported into MEGAN, using a synonym file that maps the SILVA accession numbers onto the NCBI taxonomy.

Conclusions: Environmental samples are often studied using both targeted 16S rRNA sequencing and random shotgun sequencing. Hence tools are needed that allow one to analyze both types of data together, and one such tool is MEGAN. The ideas presented in this paper are implemented in MEGAN 4, which is available from: http://www-ab.informatik.uni-tuebingen.de/software/megan.

Figure 1

A part of the final SILVA file obtained using ARB software from SILVA website. The entries are exported and used in the mapping file.

Figure 2

A part of the NCBI taxonomy file downloaded from the NCBI archive. This file is used to obtain a mapping between NCBI taxon IDs and the associated NCBI taxon names.

Figure 3

Advanced-tab in the Import from BLAST-menu for BLAST output files requiring the mapping file.

Figure 4

MEGAN4 analysis result for ‘mice-data’ against the SILVA database. Numbers in taxonomic tree indicate the number of assigned reads to each taxon collapsed at ‘genus’ level of NCBI taxonomy.

Figure 5

MEGAN 4 analysis result for ‘mice-data’ against the NCBI-NR database. Although we expected to obtain no significant hits, a wide range of hits were found. Numbers in taxonomic tree indicate the number of assigned reads to each taxon collapsed at ‘genus’ level of NCBI taxonomy.

Figure 6

Comparison of the taxonomic analysis of a 16S rRNA dataset [15], computed using six different approaches: MEGAN4’s BLASTN-based SILVA analysis, the RDP website’s classifier [6], MG-RAST’s RDP-based approach [21], the SILVA website’s aligner [5], MG-RAST’s Greengene based approach and MG-RAST’s SILVA based approach targeting the SSU gene. In this figure, the bar charts on higher-rank nodes reflect the total number of reads assigned to the corresponding node or to any of the nodes in the subtree below the node.