Earlier genomic analysis approaches; Whole genome sequencing

Question 1

Early in the 20th century…prior to the development of early methods for DNA sequencing
Geneticists typically followed a two-part classical genetics approach to identify and characterize all of the genes in an organism’s genome:

Answer 1

1. they identified spontaneous mutations or collected mutants induced by chemical or physical agents.
2. they generated linkage maps using these mutant strains.

Identified genes in model organisms like Drosophila, mice, maize, yeast, bacteria and viruses.

Question 2

The genomics era was introduced by

Answer 2

Sanger et al. when they sequenced the 5400 bp genome of the phage ɸX174 in 1977

Question 3

In the 1980s, geneticists interested in mapping human genes began using recombinant DNA technology to map DNA sequences to specific chromosomes. Most of these sequences were not actually full-length genes but marker sequences such as restriction fragment length polymorphisms (RFLPs). Once assigned to chromosomes, these were used in pedigree analysis to establish linkages between the markers and disease phenotypes for genetic disorders. This was called positional cloning and was used to map, isolate, clone, and sequence genes for cystic fibrosis, neurofibromatosis, and many other disorders.

Question 4

In the 1990s it was estimated that there were approximately 100,000 genes in the human genome which was later found to be inaccurate. Rapid advances in DNA-sequencing methods now enable sequencing larger and more complex genomes of eukaryotes, including the human genome. These DNA-sequencing technologies are responsible for modern genomic analysis.

Question 5

Many disadvantages:

Answer 5

require a mutation in a gene before a linkage map can be constructed; very slow; only in non-human organisms.

Question 6

In the 1980s, recombinant DNA technology was used to map human DNA sequences to specific chromosomes.

Answer 6

These sequences were not full-length genes but marker sequences such as restriction fragment length polymorphisms (RFLPs).
Once these markers were assigned to chromosomes, it could be used to establish linkages between the markers and disease phenotypes for genetic disorders.
Allowed for more than 3500 genes and markers to be mapped to human chromosomes.

Question 7

In the 1990s, human genes estimated at ±100 000 – impossible to map and clone using traditional methods

Question 8

Genomics allows sequencing of

Answer 8

entire genomes

Question 9

Most widely used strategy for sequencing and assembling an entire genome involves

Answer 9

variations of a method called whole-genome sequencing (WGS) or shotgun cloning.

Question 10

In whole-genome sequencing (WGS) or shotgun cloning, what are the purpose of restriction digests?

Answer 10

(or sonication) of whole chromosomes generate thousands to millions of overlapping DNA fragments.

Question 11

Whole-Genomes are Sequenced and assembled using Bioinformatic applications

Answer 11

Software that create DNA sequence alignments.
Alignments identifies overlapping sequences, which can be used to map onto chromosomes.
Overlapping sequences are adjoining that together form a continuous DNA fragment, called a contig.

Question 12

The WGS shotgun method was developed by

Answer 12

J. Craig Venter at The Institute for Genome Research (TIGR) in 1995, when they sequenced the 1.83-million-bp genome of the bacterium Haemophilus influenzae.
This was the first complete genome sequence from a free-living (i.e. nonviral) organism demonstrating “proof-of-concept” that shotgun sequencing could be used to sequence an entire genome.

Question 13

In the example, alignment software has identified an overlap between three fragments of sequenced DNA (contigs 1, 2 and 3) from human chromosome 2. The software is able to assemble the three sequences into one much larger sequence using the overlaps. In this way, the sequence of the entire chromosome can be assembled in silico.

Question 14

While possible, it is a time consuming and costly exercise to sequence an entire genome by the Sanger method.
The major technological breakthrough that made genomics possible was the advent of computer-automated sequencers (high throughput) like the 454 sequencing method.

Answer 14

High-Throughput Sequencing and Its Impact on Genomics

Question 15

Conventional sequencing is too slow for WGS…
Discovered High-Throughput Sequencing (HTS)

Answer 15

Computer-automated DNA sequencers:

Designed for high-throughput sequencing, thus making genomics possible
Essential for Human Genome Project
Sequencers contained multiple capillary gels (96)
Generated over 2 million bp per day

Question 16

Sequencing cost and time have decreased remarkably

Answer 16

The major technological breakthrough that revolutionised genomics possible was the advent of Next-Gen Sequencing (NGS), like the 454 pyrosequencing method.
Sequencing the first human genome cost about $1 billion and took 13 years to complete; today it costs < $1000 and takes a day.
Moore’s law

Question 17

Prior to the widespread use of WGS, genomes were being assembled using a

Answer 17

clone-by-clone approach, also called map-based cloning.

Question 18

Initial progress on the Human Genome Project was based on

Answer 18

clone-by-clone approach

Question 19

clone-by-clone approach steps

Answer 19

In the clone-by-clone approach, individual DNA fragments from restriction digests are aligned to create the restriction maps of a chromosome. These restriction fragments are then ligated into vectors such as bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs) to create libraries of contigs. DNA fragments in BACs and YACs can be further digested into smaller, more easily manipulated pieces that are subcloned into smaller vectors such as plasmids so that they can be sequenced in their entirety. After each sequenced fragment was analyzed for alignment overlaps, a chromosome can be assembled.

Question 20

Question 21

Compiling genome sequences

Answer 21

Draft sequences and reference genomes

Question 22

Draft sequences and reference genomes

Answer 22

First assemblies generate draft genomes.
Final assemblies constitute reference genomes.
A reference genome is never 100% final - “final” is dictated by the number of errors that are accepted as a cut-off.
Accuracy improved by sequencing multiple times.
Coverage (or depth) is the number of times that a particular nucleotide appears in the same position after multiple reads have been compiled.
Once compiled/error checked, the genome is analysed to identify:
- Gene sequences
- Regulatory elements
- Other features that reveal genetic information