12.1 Sequencing/genotyping applications

Question 1

Genome assembly by shotgun sequencing mechanism

Answer 1

Shear genomic DNA into short sequences, then sequence using next gen. Assembler software looks for overlaps to assemble larger fragments (contigs). Issue with repetitive regions - overcome with nanopore reads.

Question 2

Advantage of long reads

Answer 2

Help with assembly and alignment of shorter reads

Question 3

Genome assembly by shotgun sequencing is a big bioinformatics task

Answer 3

A genomic DNA sample contains many thousands of copies of the whole genome and each segment is sequenced hundreds-thousands of times.

Question 4

Read or sequencing depth

Answer 4

Greater read depth (the number of times a particular base is represented) gives more confidence.
First sequence - great depth.
Additional copies - lower depth is sufficient.

Question 5

Transcriptomics, gene expression analysis

Answer 5

Isolate mRNA, convert to cDNA, shear cDNA, add adaptors and sequence by next gen. Bioinformatics to sort sequences in to genes.
The number of times a gene appears in the data represents the degree to which that gene is being expressed in the sample of interest.
Next gen.

Question 6

Identifying species, DNA barcoding; the inventory of animal life

Answer 6

Many genes/DNA can be used to identify species, but mitochondrial DNA COI gene is the most widely used, uses sanger sequencing.

Question 7

DNA barcoding to identify sea foods

Answer 7

Seafood fraud is common, and endangered species are harvested. Sampling these for their “barcode’ can expose.
Sanger.

Question 8

Evolutionary relationships of living and extinct species of human

Answer 8

Targeted mtDNA (sanger) and used estimates of how fast it evolved. Count the number of mutations that seperate groups to show a common ancestor.
Sanger and next gen (for ancient DNA).

Question 9

Studying microbiomes

Answer 9

Account for most diversity on earth. Isolated DNA is amplified and sequenced using next gen to then sequence and see what species are present, and in what relative abundance.
Next gen.

Question 10

Environmental DNA

Answer 10

All organisms shed DNA that can be isolated from environmental samples then sequenced to identify who is present. Taxon specific primers allow particular species to be targeted.
Next gen.

Question 11

DNA fingerprinting (profiling)

Answer 11

First used mini satellites (10-100 bps) that are repeated many times in tandem arrays.
13 standard microsatellite loci are used in criminal forensics.

Question 12

Microsatellites

Question 13

Microsatellite genotyping PCR

Answer 13

Fluorescent primers flank the sequences, amplify products, separate by electrophoresis (codominance, detect both).
Use capillary electrophoresis machine with primers of different colours (multiplex analysis).
Bands are distinguished by time based on size.

Question 14

Microsatellite “DNA fingerprinting” in criminal forensics

Answer 14

Requires only a tiny sample of DNA, ideal for forensics.
These methods help convict and exonerate.
Methods are so sensitive though, that contamination can be a problem.

Question 15

Microsatellite/STR/SSRs in genetic disorders

Answer 15

Trinucleotide repeat loci, normal to have this microsatellite but these disease have too many repeats.
Eg. Myotonic dystrophy
Klinefelter’s syndrome
Fragile X syndrome

Question 16

Restriction enzymes to detect DNA polymorphism

Answer 16

Gain or loss can be detected using gel electrophoresis., most commonly caused by single nucleotide polymorphisms.

Question 17

Single nucleotide polymorphisms

Answer 17

Single base mutations, every 800-1000 bp. Several million SNP loci. Typically di-allelic (one of two nucleotides).
Close together due to low recombination - haplotypes.

Question 18

SNP chips

Answer 18

Microarrays that are designed to allow many SNPs to genotyped at once.
Use glass slide and DNA hybridisation-based assay for rapid screening.

Question 19

Genome-wide association (GWAS)

Answer 19

Aims to find genetic links to disease - looks for SNPs with alleles correlated with a certain trait.
BUT many diseases are influenced by many genes, and the environment.