Week 5 Flashcards
History of Sequencing: Fred Sanger
In 1977, he published the first whole genome sequence of a microorganism, bacteriophage ϕX174, using a method called dideoxy chain termination sequencing, now commonly referred to as Sanger Sequencing
History of Sequencing: Craig Venter
The Institute for Genomic Research (TIGR) completed the first genome sequence from a cellular organism, Haemophilus influenzae, in 1995 using his shotgun approach to whole genome sequencing
History of Sequencing: Jonathan Rothberg
In 2000, Rothberg founded 454 Life Sciences and brought to the market the first commercially-available machine for massively parallel DNA sequencing in 2005, a
Genome Sequencer GS20, which used a technology called pyrosequencing.
454 Life Sciences / Ion Torrent
454 Life Sciences (acquired by Roche Diagnostics) and the Baylor College of Medicine were the first to publish the whole genome sequence of a single human individual, James Watson, using massively-parallel sequencing
In 2007, Rothberg founded Ion Torrent (acquired by Thermo Fisher Scientific), which developed an ion semiconductor sequencing method and released the Personal Genome Machine (PGM) in 2011.
History of Sequencing: Klenerman & Balasubramanian
David Klenerman and Shankar Balasubramanian invented a next-generation DNA sequencing method based on the detection of fluorophore-labelled nucleotides as they are incorporated into the DNA strands.
The DNA template is cut into smaller pieces and adapters are attached to act as reference points during amplification, sequencing, and analysis
Applications of WGS in Infectious Disease Studies
Include the following:
1. Detection of antibiotic resistance mutations
2. Tracking the transmission of specific strains
3. Identifying virulence loci
Guidelines for tuberculosis control in NZ 2019
The important areas where best practice is still being defined are not covered in these guidelines - for example, whole genome sequencing, which may enable faster diagnosis of resistance and better definition of clustered cases
Tracking Transmission
Genetic distances estimated with maximum likelihood.
Each blue circle represents a node of people who were infected with isolates separated by no SNPs.
Each number within a circle is one patient, the number indicates at which year during the outbreak they were diagnosed.
Black circles are added when patients within blue circles are separated by more than one SNP; one black circle represents a difference of one SNP.
Dashed lines in clusters three and ten show larger SNP distances with numbers representing the SNP difference.
- Tracking Transmission Detailed investigation of cluster seven.
(A). Genetic tree and matrix of nucleotide variants. Genetic distances estimated with maximum likelihood. Each blue circle represents a node of people who were infected with isolates separated by no single nucleotide polymorphisms. Numbers within nodes are patient numbers and years of sample isolation are given in parentheses. Black circles are added when patients within blue circles are separated by more than one SNP. The matrix shows nucleotide variants. (B). Epidemiological network.
(C). Time of onset of symptoms, diagnosis, and treatment. Sputum smear positive samples show probable infectious periods
purpose of Fred Sangers work
From its early beginnings using Fred Sanger’s dideoxynucleotide sequencing method, whole-genome sequencing has progressed to a highly-automated, massively-parallel technique for which a number of platforms have been commercialised.
Current technologies in use include:
- Pyrosequencing (Roche)
- Ion Semiconductor sequencing (Thermo Fisher)
- Supported Oligonucleotide Ligation Detection sequencing (Thermo Fisher)
- Single Molecule Real Time sequencing (PacBio)
- Nanopore sequencing (Oxford Nanopore)
- Illumina-dye sequencing (Illumina)
bioinformatics analysis
Several online databases are available for the bioinformatics analysis of raw
FASTQ sequence files and to perform analyses that include single nucleotide polymorphism (SNP) calling, phylogenetic lineage determination, and identification of drug resistance mutations
sensitivity and specificity of whole genome sequencing
Work is ongoing internationally to validate the sensitivity and specificity of whole genome sequencing for routine use in medical laboratory diagnostics of infectious diseases such as tuberculosis
