Lecture 16- Genome sequencing

Question 1

sanger sequencing process

Answer 1

different ddNTPs- fluorescently labelled, each halted strand separated be electrophoresis, can then read the sequence

Question 2

sanger sequencing pros

Answer 2

accurate and easy to perform
one sequence at a time- so labour intensive, can get expensive, slow

Question 3

next gen sequencing process

Answer 3

uses fluorescence emitted by the DNA extension reaction

Question 4

pros of NGS

Answer 4

relatively low reagent volumes needed
can sequence multiple sequences in parallel- more effective process
however accuracy is lower

Question 5

how does illumina sequencing work

Answer 5

adaptors on a glass slide, ‘bridges’ form, dyes are added and can then see what bases are being added

Question 6

pros of illumina

Answer 6

can produce huge volumes of reads- up to 1tb per day

Question 7

benefits of genome re-sequencing

Answer 7

can look at genetic variation within a population or species- often done w humans to look at SNP and indel locations etc

Question 8

how can you look at the chromatin configuration of a genome

Answer 8

chromosome conformation capture- looks at the interactions between parts of DNA by cutting the DNA strand, and ligating the strands that remained due to being attached to ribosomes etc
can then sequence and map these

Question 9

how do you identify replication origins from sequencing data

Answer 9

looking at where there is more representation, can see ‘peaks’ in copy number at the replication origins

Question 10

how can sequencing data be used to look at the transcriptome

Answer 10

sequencing mRNA at specific points

Question 11

ATAC-seq

Answer 11

Assay of transposon accessibility of chromatin - kinda what it sounds like, sequencing exposed DNA and mapping it to the genome to see where is being transcribed and where is repressed

Question 12

how can you detect methylation

Answer 12

switching all unmodified cysteines to uracil- therefore only methylated cysteine remains

Question 13

ChIP-seq

Answer 13

Chromatin immunoprecipitation followed by sequencing- cross-linking proteins to DNA, removing other DNA, precipitation, purification, sequencing

Question 14

oxford nanopore

Answer 14

more portable technology, third-generation sequencing using long reads, makes de novo assembly easier

Question 15

example of a long-read sequencing technology

Answer 15

PacBio- generally longer reads than nanopore