metabarcoding Flashcards

1
Q

what is DNA barcoding

A

DNA barcoding = Molecular taxonomic identification using short DNA sections (barcode) from part of a genome - allows species identification
- sampling -> DNA extraction -> PCR -> Sanger sequencing -> database alignment / bioinformatics

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2
Q

why do we extract DNA and what are the challenges

A
  • Optimise method based on sample type maximising DNA concentration & quality
    Challenges:
    >Physically and/or chemically difficult
    >Extraction biases between different organisms
    >DNA degradation
    >Low DNA concentrations
    >Contamination
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3
Q

how can we apply DNA barcoding

A
  • detect rare+ invasive species
  • response to pollution, warming, acidification
  • biodiversity monitoring
  • ecosystem change over time
  • diet analysis
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4
Q

difference between barcoding and metabarcoding

A
  • barcoding = Targeted sample identification (one species at a time). -tend to use Sanger sequencing
  • metabarcoding = Profiling complex communities (multiple different species at same time) - tend to use high-throughput sequencing e.g. NGS, 3rd genetic sequencing
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5
Q

what’s important when Selecting good genomic regions for metabarcoding during PCR

A
  • good databases for identification
  • sufficient variability between species/strains
  • short length (~150-300 bp); good for NGS & DNA quality
  • conserved flanking sites for universal primers
  • Primer selection depends on the question
  • Common barcode regions include = COI in mitochondrial genome (vertebrates), 16S (prokaryotes), ITS (fungi), RuBisCo (plants)
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6
Q

what are the common barcode regions for vertebrates, prokaryotes, fungi and plants

A
  • COI in mitochondrial genome (vertebrates)
  • 16S (prokaryotes)
  • ITS (fungi)
  • RuBisCo (plants)
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7
Q

High-throughput sequencing methods

A
  • 1st gen sequencing (sanger sequencing) = 1 thing at a time
  • NGS = multiple at same time
    >Millions of short reads
  • 3rd gen sequencing (oxford nanopore, PacBio SMRTcell)
    >Ultra-long reads
    >More accurate
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8
Q

what happens during PCR before sequencing

A

PCR amplicons from each individual sample are tagged using unique index adaptor - allows 100’s of samples to be multiplexed and sequenced simultaneous

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9
Q

what’s included in the bioinformatics part of analysis

A
  • filtering of sequence reads - cluster ‘good’ reads into taxonomic units i.e. OTUs (97%) or ASVs (100%) to represent distinct taxa
  • Align reads to reference databases & annotate e.g. BLAST - helps identify the taxa represented in each cluster
  • Quantify reads for each taxonomic unit - estimate the abundance of each identified taxonomic unit in the sample
  • Can then look at community diversity / structure
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10
Q

positives for MtDNA as a marker

A
  • single copy - easy to sequence - Sanger Sequencing
  • recovery from very small or degraded biological
    samples = higher than nDNA - mtDNA molecules exist in thousands of copies per cell
  • COI used for barcoding other useful genes Cyt (also barcoding), 16S, Control Region (or D-Loop)
  • Conserved arrangement of genes - easier to design universal primers
  • mtDNA has higher rates of mutation - more likely to exhibit sequence variation
  • mtDNA is maternally inherited - useful for studying sex-biased population processes and hybridisation between differing populations
  • no recombination - easier to trace genetic lineages and so reconstruct historical processes
  • mtDNA exhibits a small effective population size (¼ of nuclear) more susceptible to genetic drift and readily displays demographic changes in populations (such as past bottlenecks)
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11
Q

negatives for MtDNA as a marker

A
  • mtDNA acts effectively as a single locus and cannot compare genealogies of multiple independent loci
  • small Ne may exaggerate historical events and underestimate genetic diversity
  • Only provides a genetic picture of female-linked population processes in the evolution of the species
  • the selective neutrality of mitochondrial genes has been called into question could also be involved in speciation
  • mtDNA sequences have been found in nuclear DNA, known as pseudo- mtDNA or nu-mtDNA (so violating most of the positives)
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