Metagenomics and Metatranscriptomics Jinyu Yang Bioinformatics and Mathematical Biosciences Lab July/15th/2016 Background Conventional sequencing begins with a culture of identical cells as a source of DNA Bottleneck Vast majority of microbial biodiversity had been missed by cultivation-based methods Large groups of microorganisms cannot be cultured and thus cannot be sequenced Bottleneck For example: 16S ribosomal RNA sequences 1. Relatively short 2. Often conserved within a species 3. Generally different between species Many 16S rRNA sequences have been found which do not belong to any known cultured species there are numerous non-isolated organisms Cultivation-based methods find < 1% of the bacterial and archaeal species in a sample Next-generation sequencing The price of DNA sequencing continues to fall All genes from all the members of the communities in environmental samples Metagenomics The term “Metagenomics” first appeared in 1998 Metagenomics: 1. genomic analysis of microbial DNA, which is extracted directly from communities in environmental samples 2. Alleviating the need for isolation and lab cultivation of individual species 3. Focus on microbial communities Metagenomics Taxonomic analysis (“who is out there?”) Assign each read to a taxonomy Functional analysis (“what are they doing?”) Map each read to a functional role/pathway Comparative analysis (“how do different samples compare?”) Level 1: sequence composition Level 2: taxonomic diversity Level 3: functional complement, etc. Metagenomics Metagenomics Metagenomics Microbial Communities Microbial Communities Metagenomics Metatranscriptomics Entire genetic complements including Subset of genes that are transcribed phylogenetic and functional genes from active population DNA-based mRNA-based Microbial Communities protein-based Metabolomics Metaproteomics Global metabolic profiles produced Proteins that are ultimately expressed from community function by gene regulations Metatranscriptomics Functional diversity Metagenomics Metabolic diversity Which genes are expressed? Metagenomic studies provided a snapshot of the genetic composition of the community Metatranscriptomics Metatranscriptomics is the study of the function and activity of the complete set of transcripts (RNA-seq) from environmental samples. Metatranscriptomics: profiling of community-wide gene expression (RNA-seq) Metatranscriptomics Gene activity diversity Gene expression abundance Differential gene expression analysis Metatranscriptomics Gene activity diversity How many different genes are expressed in a microbial community across all species? How is the functional and pathway diversity? Metatranscriptomics Gene expression abundance Which are the highest expressed genes in a specific environmental condition? What is the most important functionality (pathway) needed in an environment? Metatranscriptomics Differential gene expression analysis Which genes show a highest change in expression levels between different conditions (biomarker detection)? Metatranscriptomics Raw RNA-seq reads Low quality trimming Preprocessing rRNA identification and removal Dereplication Annotation and Assembly Gene activity diversity Gene expression abundance Analysis Differential expression Thanks!.