9 - Multi-omics Approaches for Microbial Communities Flashcards
Why is AMR difficult to trace
- It affects every environment on earth
- It involves every kingdom of life
- Not just one factor (multitude of different mechanism and any single microbe will have more than one mechanism of AMR)
Extracting nucleic acid from an environmental sample
- Can capture DNA from non culturable organisms
- Conclude which organisms are predominant by analyzing genomes
- Can detect lateral gene transfer
- Build phylogenies using DNA
- Compare diversity
Three popular -omics methods
- Targeted metagenomics
- Metagenomics
- Metatranscriptomics
Targeted metagenomics
- 16S rRNA gene profiling
- Quantitative method for determining population diversity and relative prevalence of members in a community
Metagenomics
- Not quantitative
- Used for functional genomics
Functional genomics
- Metabolism
- Antibiotic resistance markers
- New synthetic pathways for novel compounds
- Finding new microbes
Metatranscriptomics
Quantitative analysis of RNA to identify active metabolic pathways
Illumina sequencing steps
- Prepare genomic DNA sample
- Attach DNA to surface
- Bridge amplification
- Fragments become double stranded
- Denature the double stranded molecules
- Complete amplification
Building a sequence of an amplicon
- Clusters are converted to single strands
- DNA polymerase is used to synthesis a second strand using nucleotides each with a different coloured fluorophore
- After each base is added the fluorescence is measured as the next base is added sequentially.
- This generates the sequence
- Allows 1000,000s of clusters to be sequenced in parallel
- The adaptors contain unique barcodes that enable the
distinction of each amplicon by computer algorithms
Steps of DNA sequence assembly
- Base calling
- Trimming
- Assembly
Base calling
- Conversion of the electronic signal to the reported base call
- Quality control threshold and if this is not met the base will be termed unknown
- Quality control removes noise from data leaving only high quality base calls
Trimming
- Primer sequences are removed leaving the target sequences
- This cleaned data goes into assembly pipelines
Assembly
- Illumina generates “pair-end reads” ie. Each 330-440 bp fragment is sequenced from both ends sequentially
- Computer Algorithms are used to identify the paired adaptors in each amplicon to
generate a consensus sequence of both strands for each 440 bp fragment (called a
kmer)
Kmer sequences
- Can be aligned to create the genome sequence
- Either de novo when the genome is unknown OR
- Aligned or mapped to a reference genome
Metagenomics steps
- Extract total DNA
- DNA is fractionated (no amplification of a particular gene)
- Sequence
- Bioinformatics
Bioinformatics
- Sequences are assembled into contigs
- Binning separates contigs into genomes of known bacteria / archea / virus
- Use a series of computational and statistical methods to assure sufficient coverage by assessing marker genes
- Organise the genes into metabolic pathways (crosscheck for characteristics of known genera)
Metatranscriptomics steps
- Remove DNA
- Purify the RNA
- Convert RNA sequence to DNA sequence using a reverse transcriptase to create a library of copy DNA (cDNA)
- Sequence
- Bioinformatics analysis of sequence identity and the number of identical reads are counted
High counts in metatranscriptomics
More transcription
Low counts in metatranscriptomics
Less gene transcription
Pros of metatranscriptomics
overview of the entire microbial population
Cons of metatranscriptomics
- Cannot tell what individual strains/species are carrying
- e.g. g. Horizontal gene elements may appear multiple times but can’t tell if they are in the same strain or not
Non culture dependent approaches for linking horizontal gene elements to a bacterial genome
Proximeta
Proximeta
- Type of proximity-ligation sequencing that permits reconstruction of high quality metagenomes of microbial communities, and also matches mobile genetic elements to their host genomes
- Before DNA extraction, the bacterial cells are permabilised and the nucleic acid is cross-linked together (thus capturing the plasmids/phages etc)
- After extraction the DNA is fragmented using endonucleases, then
biotinylated and ligate cross-linked fragments so that chromosome and plasmid regions once linked by proximity are now joined on the same DNA strand for NGS. - The bioinformatics pipeline identifies the species and plots the elements against the
phylogenetic tree.
Pros of proximeta
Shows association of antibiotic resistance mobile elements to a particular family (eg. Neisseriaceae)
Cons of proximeta
- Not strain specific
- Do not know if the genes are expressed unless transcriptomics is performed or the strains are cultured
Actinobacteria enrichment
Enriched for phosphotransferases that modify aminoglycosides and beta-lactamases that degrade beta-lactams
Firmicutes enrichment
Enriched for MFS pumps,
most antibiotic modifying enzymes and target protection mechanisms of resistance
Proteobacteria enrichment
Enriched for RND and other efflux pumps in addition to genes modulating resistance.
Human microbiome enrichment
enriched in most mechanisms
of resistance
Resistomes governed by ecology
A few AR mechanisms were shared between the human microbiome and soil, but most diversity was dependent upon the phyla in each ecological niche
Technological advances in computing and sequencing
- New sequencing technologies (Illumina high through put sequencing)
- Computing power (New computing algorithms to analyse huge datasets)
