Add Introduction to Metagenomics lecture notes
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* Metagenomics applied to surveillance of pathogens and antimicrobial resistance
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** Week 1
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*** Introduction to Metagenomics
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In order to fight against antimicrobial resistance, we need to know where the pathogens and the transmisison routes are.
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Metagenomics allows us to identify individuals in a complex sample, using DNA sequences. It is a sequencing-based analysis of genomes contained in an environmental sample.
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We need to isolate the genomes of the microbes, the same applies to all microbial genomics techniques, which we'll compare to metagenomics:
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**** Microbial genomics
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***** 16S rRNA profiling
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We amplify the 16S ribosomal RNA obtaining as a result an idea of the taxonomic composition of the sample. The 16S analysis offers deep insight, such as presence of low abundance organisms.
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Metagenomics is a more generic approach, by randomly amplifying whole genomes we can learn more about the function.
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***** Whole Genome Sequencing (WGS)
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We isolate and cultivate specific microbes from a complex sample. We obtain taxonomic identity information and specific information such as virulence, antimicrobial resistance, etc.
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***** Single Cell Sequencing
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It has the same principles as the previous method and it's used when it's not possible to cultivate the microbe.
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**** Metagenomics project
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A metagenomics project involves many steps, we will group them in 2 categories:
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***** Laboratory steps
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1. DNA/RNA isolation: RNA is translated to cDNA
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2. Fragmentation: current next generation sequencing methods cannot sequence long fragments (exception: Nanopore sequencing)
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3. DNA sequencing: we obtain files with the sequence and quality information
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***** Data analysis
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- Reads: we analyze directly
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- Binning and assembly
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**** Current challenges
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Metagenomics still faces some challenges, mostly due to the sequencing process:
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- Sequencing data can belong to the tip of the iceberg (for complex samples)
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- Background DNA sequences can make up a considerable percentage of the data (e.g. host environment)
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- Lack of reference genomes due to novel organisms, although some methods exist to allow us to identify them
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- Low-quality reference genome (contaminant sequences -> false-positive)
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- Quantification of the pathogens is difficult due to the different genome sizes
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- Assembly of reads from different organisms (chimera formation)
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- Unknown function of novel proteins
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