Workshop 1 - Sequencing Flashcards
what are the three forms of sequencing?
Sanger First Generation Sequencing
Next Generation Sequencing (Illumina/Iontorrent)
Third Generation Sequencing (Nanopore/PacBio)
average reads for the three types of sequences:
Sanger First Generation - 800bp
Illuminati/Iontorrent Next Generation - 150bo
Nanopore/PacBio Third Generation - >3000bp
First Generation Sanger Sequencing Summary:
- provides only the sequence
- one gene or transcript at a time
- have to select gene to sequence
- cheap per reaction
- time consuming
Sanger First Generation (Termination) Sequencing Summary:
- average read 800bp
- provides only the sequence
- one gene or transcript at a time
- have to select gene to sequence
- cheap per reaction
- time consuming
Illumina/Iontorrent Next Generation Summary:
- multiple genes or transcripts at a time
- broad and non specific
- can be used for differential expression
- requires fragmentation - break long transcripts into smaller pieces
- reliant in bioinformatics to piece it back together
Nanopore/PacBio Third Generation Summary:
- multiple reads or transcripts at a time
- broad and non specific
- can be used for differential expression
- sequences entire transcript
- currently less accurate
chain-termination (sanger) sequencing provides the sequence from a…
defined starting point
in sanger sequencing, extension of the DNA products continues until…
the incorporation of a nucleotide with a fluorescent molecule
what is separation based on within the Sanger Termination Sequencing?
based on size occurs and a laser
detects the fluorescent molecule to “read” the sequence
why is there a limit to how long the DNA products can get (approx. 1000 bp) in Sanger Chain-Termination Sequencing?
due to the limited amount of nucleotides with and without fluorescence
what are Sanger/Chain-Termination Sequencing methods ideal for?
ideal for sequencing short stretches of plasmid or PCR product
Sanger/Chain-Termination Sequencing Price:
reaction is £5 at most so good for a
single sequence
sequencing by synthesis takes the concept from chain-termination (sanger) sequencing and enhances it by:
by allowing the removal of the fluorescent molecule to allow additional nucleotides to be added
how are millions of DNA sequences read simultaneously in next-generation sequencing?
using high-resolution cameras, millions of DNA sequences to be read simultaneously
sequences no longer than 300bp can be generated in next generation sequencing which is shorter than most genes so…
computationally we are able to match and join them to get more complete sequences, and most cases we can identify a gene from these short reads
what’s so good about next generation sequencing?
it is the ‘gold-standard’ as accuracy is usually excelled across the whole read
can multiplex and get a large number of samples sequenced
The latest version of sequencing use a change in the current generated across a membrane to figure out what the sequence is:
Nanopore/Long-Read Sequencing
how does detection work in Nanopore/Long-Read Sequencing?
As the nucleotides have different structures, they block the flow of ions through the pore in different ways which can be detected and
correlated to one of the bases
what sort of readings does Nanopore/Long-Read Sequencing do?
very long reads in the 1000’s of
bases and can deal with repetitive sequences
what sort of readings do you achieve with Nanopore/Long-Read sequencing:
very long reads in the 1000’s of
bases and can deal with repetitive sequences
downside to Nanopore/Long-Read Sequencing:
The accuracy is not as good as other
methods, so there is significant reliance on consensus alignment
consensus of Long-Read/Nanopore Sequencing:
Consensus is where you take multiple
reads that align together and taking what is common across all the reads for the true sequence
why apply drug treatment and fund drug development (development)?
Finding new genes involved may help identify new target genes and pathways to develop treatments against
why bother with genetic testing?
allows us to identify genes that may underlie heritable forms of disease, and highlight those where mutations could be passed on to cause disease in future generations
allows for treatments to be provided to those with increased risk to reduce this risk
genetic testing:
- where one is tested to see wether mutations exist in their DNA for the disease
- often sample taken via cheek swab or blood
- commercial kits available but these are often limited in what they look at due to the sensitive nature of genetic testing
- in clinical practice, this sequencing is targeted to only genes
that are known to be associated with the disease
genetic testing:
With the sequencing results looking for a mutation in a gene, we have to think carefully about how
we interpret and use this information
This is the same whether we are doing genetic testing or for looking to identify a mutation for research
• We need to consider the change in the sequence we find, but also the context of the study and the
context of the person
• There is not always a simple and straightforward answer to these questions
genetic testing:
This is where someone is tested to see whether they have
mutations in their DNA for the disease
Often this involves taking blood or a cheek swab to get DNA material which is then sequenced
There are commercial kits that are available but often these are limited in what they look at due to the sensitive nature of genetic testing
In clinical practice, this sequencing is targeted to only genes
that are known to be associated with the disease
assessing the results of genetic testing:
With the sequencing results looking for a mutation in a gene, we have to think carefully about how
we interpret and use this information
– This is the same whether we are doing genetic testing or for looking to identify a mutation for
research
• We need to consider the change in the sequence we find, but also the context of the study and the
context of the person
• There is not always a simple and straightforward answer to these questions
APOE4:
APOE4 is a well-established genetic risk factor for age-related cognitive decline and Alzheimer’s disease
• Generally, people with APOE4 tend to have memory issues compared to those that don’t
• A study on 1770 Americans found white Americans with APOE4 had decreasing performance in a verbal memory task while African Americans showed no impairment
• APOE4 may have different effects on
different genetic backgrounds as a result of interactions with other alleles of genes
