week 9

Question 1

what are microsatellites

Answer 1

• repeat sequences
• nuclear DNA
• rapidly evolving
• biparentally inherited
• polymorphic: many alleles per locus
• selectively neutral

Question 2

what are SNPs

Answer 2

• variation in the genome; DNA profile of an individual
• nuclear DNA
• point mutations
• biparentally inherited, 3 possible combinations : heterozygous GA, homozygous GG, homozygous AA

Question 3

describe the SNP scoring procedure

Answer 3

1. sample DNA is added to mix containing allele specific SNP-primer, Taq polymerase and fluorescent reporting system
2. during real time PCR denaturation, heat separates DNA into single strands. SNP-primer binds to target SNP region and amplifies
3. polymerase amplifies the region marked by SNP-primer (PCT cycles repeated = exponential replication)
4. fluorescence system binds to amplified regions, releasing a signal

Question 4

what is the difference between sanger sequencing and next generation sequencing

Answer 4

sanger sequencing:
- slow
- cheap for a small number of genes/samples
NGS:
- more expensive
- but only if a small number of genes/samples
- 1000s of samples analysed simultaneously: FAST!

Question 5

what is metabarcoding

Answer 5

• sequencing millions of copies of a specific target region from a mixed slurry of material
• one gene in many genomes

Question 6

describe the metabarcoding approach

Answer 6

• DNA extraction -> PCR -> sequencing -> bioinformatics
• replicated sampling design to capture diversity
• technical replicates; independent DNA extractions and PCR amplifications

Question 7

barcoding vs metabarcoding

Answer 7

• DNA fragment size - b=usually >500bp mb=<400bp(degraded DNA)
• specificity - b=taxon level mb=taxon (single primer pair) to multi-taxon (multi-primer pairs)
• versatility - b=not required beyond taxon of interest mb=high versatility to amplify exhaustively all target groups
• taxon resolution - b=species level mb=ideally species; otherwise MOTUs
• taxon database - b=major goal mb=desirable but not often available

Question 8

challenges of sequencing eDNA

Answer 8

• degraded
• contamination
• representation of different species
• choice of primers
• taxon bias in PCR?
• qualitative
• quantitative?

Question 9

pros of biodiversity and species ID

Answer 9

• elusive organisms
• non-invasive
• detect cryptic species
• traces in environment - faeces, hair, skin
• longevity: weeks in rivers to 1000s of years in permafrost
• non-specialist: citizen science
• cheap