ORB's comprehensive metatranscriptome analysis pipeline powers studies of diverse microbial populations based on RNA sequencing from biospecimens and environmental samples. ORB’s computational and statistical strategies include targeted genome alignment, taxonomic classification, and de novo transcriptome assembly. These research tools provide an unprecedented opportunity to examine gene regulation for many microbial species simultaneously and determination of which genes encoded in a metagenome are actually transcribed. Each of ORB’s metatranscriptome bioinformatic service packages can be combined with analysis of human epithelial cell transcripts to enhance the understanding of a host—microbiome relationship. Contact us with your unique microbiome research objectives to determine which strategies or combination of analyses will be most appropriate for your study!
This straightforward approach aligns sequencing reads to a bacterial, fungal or viral reference genome specific to the research project, e.g. 116 bacterial strains from 47 species representing the metatranscriptome of human stool samples, see Figure 1. ORB provides complementary consultations to determine which reference genome would be most useful for a specific research project. Advantages of targeted alignment include minimized risk of mapping to unintended species, a simpler analysis for ease of interpretation, and provision of detailed and specific annotation for mapped genes.
To enable ultrafast and highly accurate assignment of taxonomic labels to metagenomic DNA sequences, ORB utilizes the Kraken taxonomic classification system. Kraken is superior to earlier classification programs in terms of speed with classification of 100 base pair reads at a rate of over 4.1 million reads per minute; 909 times faster than Megablast and 11 times faster than the abundance estimation program MetaPhlAn. Furthermore, with regard to accuracy, Kraken is comparable to the fastest BLAST program. This high speed and accuracy are achievable through the examination k-mers within a read. Each k-mer is searched against a database that maps k-mers to the least common ancestor (LCA) of all organisms whose genomes contain the k-mer. A score is calculated based on how the k-mers in a sequencing read mapped to leaves in the general taxonomy tree and the read is assigned to the taxon with the highest score1.The database of k-mers to LCAs is built using all the genomes from ogranisms in the RefSeq bacterial, viral, and archaea domains. ORB harnesses Kraken’s novel algorithm to process the disparate results returned by NCBI taxonomic information and genomes for the bacterial, archaeal, and viral domains. See Figure 2 for an interactive visualization of Kraken taxonomic assignment output.
De Novo Transcriptome Assembly with Trinity
Contact us to discuss your metatranscriptomics project and how Ocean Ridge Biosciences can help you achieve your research goals!
- Wood, D. E., & Salzberg, S. L. (2014). Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome biology, 15(3), R46.
- Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. (2011) [Trinity] Full-length transcriptome assembly from RNA-Seq data without a reference genome. Biotechnology 29, 644- 652.