Lecture 2 - Sequencing and Genomics

Question 1

How many gigabases is the human haploid genome?

Answer 1

3.2 (3,200,000,000)

Question 2

Why should we care about genomics?

Answer 2

-Production animal breeding
-Understanding biology of disease
-Identification of pathogens
-Development of drugs and vaccines
-Conservation of species

Question 3

What is a genome?

Answer 3

-The complete genetic material of an organism
-Includes both genes and non-coding sequences
-In animals, bacteria and SOME viruses, it is DNA
-For many viruses it is RNA

Question 4

What percentage of the mammalian genome is protein coding genes?

Answer 4

1.5%

Question 5

What was the first type of sequencing developed?

Answer 5

Sanger sequencing

Question 6

What is Sanger sequencing?

Answer 6

Dideoxynucleotides (ddNTP’s) are added to single stranded DNA with a distinct fluorescent probe on each base. When the ddNTP reaches its compliment base pair (ex A->T), elongation stops. Each fragment is ran through gel and separated by size. Laser excitation makes the fluorescent probe visible, and can be read to see which base it is. The DNA sequence will be the compliment base.

Question 7

How many base pairs does Sanger sequencing usually yield at a time?

Answer 7

500-1000 bp

Question 8

What is the bacterial artificial chromosome (BAC) used for?

Answer 8

Used for amplifying DNA by inserting a fragment into a bacterium, where it will incorporate the fragment into its plasmid and replicate it. The product can then be isolated and used in sequencing.

Question 9

What is sequencing by synthesis (SBS)?

Answer 9

DNA polymerases and dNTPs are used to replicate the strand to be sequenced. Nucleotides are introduced with a complimentary fluorescent probe so that the base type of the incorporated nucleotide can be recognized as the DNA molecule extends. The output is read by a machine and sequenced.

Question 10

What is the difference between 1st and 2nd generation sequencing and 3rd gen?

Answer 10

1st and 2nd read small fragments of DNA, while 3rd aims to read the entire chromosomes in one go

Question 11

How does the Oxford Nanopore sequence DNA

Answer 11

Single stranded DNA moves through a pore via electrical current, and each nucleotide affects the current in a specific way. The change in current can be interpreted by minION sequencing and related to the corresponding nucleotide

Question 12

What is the issue with the Oxford Nanopore sequencing?

Answer 12

It can’t tell how many of the same nucleotide in a row there are (ex. 4 C’s in a row vs 6 C’s in a row)

Question 13

What is the SmidgION?

Answer 13

On site rapid DNA analysis. Not super accurate

Question 14

What are the pros and cons of Sanger sequencing?

Answer 14

Pros: 99.99% accuracy, cheap with only a few sequences
Cons: Short reads (500-1000bp), low output, not portable

Question 15

What are the pros and cons of Illumina (SBS)?

Answer 15

Pros: 99.99% accurate, cheap, high output
Cons: Short reads (100-300bp), not portable

Question 16

What are the pros and cons of Oxford Nanopore?

Answer 16

Pros: Very long reads (10,000-300,000bp), cheap, portable
Cons: High error rate (~8%)

Question 17

What percent of the mammalian genome is repetitive?

Answer 17

~50%

Question 18

What makes the mammalian genome repetitive?

Answer 18

-Transposons
-Simple repeats
-Multi-copy genes

Question 19

Why can repetitive DNA be an issue in sequencing and what is the solution?

Answer 19

Repetitive DNA makes it difficult to assemble the true sequence, but we can use the differing surrounding sequences to place them more accurately

Question 20

What is genome polishing?

Answer 20

Repairing errors in a sequenced genome (don’t ask me how)

Question 21

What can be used to annotate the sequenced genome?

Answer 21

RNA sequencing from target species to determine which sequences are exons (coding)