metabarcoding Flashcards
what is DNA barcoding
molecular taxonomic identification using short DNA sections (barcodes)
what is the goal of metabarcoding
profile complex communities
what is the metabarcoding pipeline (steps)
- sampling
- dna extraction
- PCR
- high-throughput sequencing
- bioinformatics
3 important things that must be addressed during sampling for metabarcoding
what, where, how much do we sample
how do we avoid contamination
how do we preserve, store, transport the sample
5 challenges of DNA extraction for metabarcoding
physically / chemically difficult,
low DNA concentrations,
DNA degradation,
extraction biases between different organisms,
contamination
give an example of when DNA extraction is physically / chemically difficult
slugs which secrete a lot of mucous
give an example of when low DNA concentrations makes DNA extraction difficult
wood
what are features of a good genomic region for metabarcoding
some conserved sites for universal primer attachment
sufficient variability in sequence
short length for NGS
good databases
what is the common barcode region for vertebrates
COI
what is the common barcode region for prokaryotes
16S
what is the common barcode region for fungi
ITS
what is the common barcode region for plants
RuBisCo
what is the difference between 1st gen, NGS and 3rd gen sequencing (what do they sequence)
1st (sanger) = single molecule sequencing
NGS = parallel sequencing
3rd = single molecule sequencing, long reads
what is involved in the bioinformatics step of metabarcoding
quality filtering of sequence reads,
cluster ‘good’ reads into taxonomic units,
align reads to databases and annotate,
quantify reads for each taxonomic unit
what does metabarcoding look at
community diversity and community structure
