DNA Sequencing Applications

Question 1

Diagnosis

Answer 1

• Sanger, next-gen
• sequencing can tell us the particular strain of virus from an infected patient
• Eg. Avian influenza strains were identified with Sanger sequencing and next-gen in china
• 800 human cases of avian flu reported since 2013
• sequence analysis shows a higher than normal mutation rate in human hosts that resulted in higher pathogenicity

Question 2

Genome assembly by shotgun sequencing

Answer 2

• next-gen and 3rd gen
• shear genomic DNA into short sequences
• seuqnce by next-gen
• assembler software looks for sequence overlaps between fragments to assemble them into larger fragments (contigs)
• now preferred way of sequencing genomes, but has problems with repetitive DNA sequences
  
  • long-read sequences used to overcome this problem

Question 3

Transcriptonomics

Answer 3

• next-gen
• isolate mRNA
• convert to cDNA
• shear cDNA, add adaptors
• sequence by next gen
• bioinformatics software sorts sequences into different types of genes
• the number of times each gene appears in the sequence data is a measure of the degree to which that gene was being expressed in the organism/tissue being studied

Question 4

Genome assembly using mapped DNA sequences

Answer 4

1. Partial digestion of DNA results in overlapping fragments that are then cloned in bacteria
2. These large insert clones are analyses for markers or overlapping restriction sites which allows the large insert clones to be assembles into a contig (continuous stretch of DNA)
3. A subset of overlapping clones that efficiently covers the entire chromosome are selected and fractures
   
   • these pieces are then cloned
4. Each of these small-insert clones is sequenced and overlap in sequence is used to assemble them in the correct order
5. The final sequence is assembled by putting together the sequences of the large clones and filling any gaps

Question 5

Determining evolutionary relationships of extinct species

Answer 5

• next gen
• extinct straight-tucked elephants one lived over wide area of Eurasia but went extinct 30,000 years ago
• thought to be closely related to Asian elephant
• DNA recovered from bones and sequenced using next-gen show they were actually more closely related to the African forest elephants
• extinct mammoths were related to Asian elephants

Question 6

Determining the evolutionary relationships of living and extinct species

Answer 6

• Sanger, next-gen
• mitochondrial DNA extracted from bones of extinct cave bears showed that they were a sister species to a living brown/grizzly bear
• brown/grizzly bears belong to 2 lineages (east and west)
• based on their mtDNA sequence, polar bears belong to a linear closely related to west brown bears

Question 7

Determining evolutionary origins and movements of modern humans

Answer 7

• next gen
• hybridization of Neanderthals and Homo sapiens
• denisovans: another species of humans
• denisovans introversion with SE asians and Oceania; high altitude adaption in tibetans
• movement of humans back to Africa from Eurasia
• modern European populations derived from 3 ancestral populations

Question 8

Identifying species

Answer 8

• DNA barcoding; the inventory of animal life
• Sanger
• using DNA barcoding to identify seafoods

Question 9

Studying microbiomes

Answer 9

• next-gen
• 90% of cells in humans are non-human
• microbiomes account for most of biodiversity on eart yet most species unknown to science
• also a huge fraction of biomass (98% of biomass in ocean is microbial)

Question 10

Influence of sex, handedness, and washing on the diversity of hand surface bacteria

Answer 10

• sampled 102 hands
• amplified, sequenced bacterial 16S rRNA genes
• phylotype defined as 97% sea identity
• typical hand >150 unique phylotypes (species)
• 4,742 species total
• only 5 common to all hands
• 3x more diversity on hands than forearm or elbow
• greater diversity on female hands
• handwashing had little effect on total species number

Question 11

Detecting species in aquatic environment

Answer 11

• next-gen & qPCR
• allow biologists to process water samples when surveying target organisms
• organisms constantly releasing trace amounts of DNA into environment
• reliable tool for aquatic species even when DNA present in low concentration
• useful for assessing species distribution or detecting rare species
• steps: filter water, extract DNA on filter, amplify mtDNA using primers, sequence (next-gen) for universal or qPCR for species specific)