Workshop 1 - Sequencing

Question 1

what are the three forms of sequencing?

Answer 1

Sanger First Generation Sequencing

Next Generation Sequencing (Illumina/Iontorrent)

Third Generation Sequencing (Nanopore/PacBio)

Question 2

average reads for the three types of sequences:

Answer 2

Sanger First Generation - 800bp

Illuminati/Iontorrent Next Generation - 150bo

Nanopore/PacBio Third Generation - >3000bp

Question 3

First Generation Sanger Sequencing Summary:

Answer 3

• provides only the sequence
• one gene or transcript at a time
• have to select gene to sequence
• cheap per reaction
• time consuming

Question 4

Sanger First Generation (Termination) Sequencing Summary:

Answer 4

• 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

Question 5

Illumina/Iontorrent Next Generation Summary:

Answer 5

• 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

Question 6

Nanopore/PacBio Third Generation Summary:

Answer 6

• multiple reads or transcripts at a time
• broad and non specific
• can be used for differential expression
• sequences entire transcript
• currently less accurate

Question 7

chain-termination (sanger) sequencing provides the sequence from a…

Answer 7

defined starting point

Question 8

in sanger sequencing, extension of the DNA products continues until…

Answer 8

the incorporation of a nucleotide with a fluorescent molecule

Question 9

what is separation based on within the Sanger Termination Sequencing?

Answer 9

based on size occurs and a laser
detects the fluorescent molecule to “read” the sequence

Question 10

why is there a limit to how long the DNA products can get (approx. 1000 bp) in Sanger Chain-Termination Sequencing?

Answer 10

due to the limited amount of nucleotides with and without fluorescence

Question 11

what are Sanger/Chain-Termination Sequencing methods ideal for?

Answer 11

ideal for sequencing short stretches of plasmid or PCR product

Question 12

Sanger/Chain-Termination Sequencing Price:

Answer 12

reaction is £5 at most so good for a
single sequence

Question 13

sequencing by synthesis takes the concept from chain-termination (sanger) sequencing and enhances it by:

Answer 13

by allowing the removal of the fluorescent molecule to allow additional nucleotides to be added

Question 14

how are millions of DNA sequences read simultaneously in next-generation sequencing?

Answer 14

using high-resolution cameras, millions of DNA sequences to be read simultaneously

Question 15

sequences no longer than 300bp can be generated in next generation sequencing which is shorter than most genes so…

Answer 15

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

Question 16

what’s so good about next generation sequencing?

Answer 16

it is the ‘gold-standard’ as accuracy is usually excelled across the whole read

can multiplex and get a large number of samples sequenced

Question 17

The latest version of sequencing use a change in the current generated across a membrane to figure out what the sequence is:

Answer 17

Nanopore/Long-Read Sequencing

Question 18

how does detection work in Nanopore/Long-Read Sequencing?

Answer 18

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

Question 19

what sort of readings does Nanopore/Long-Read Sequencing do?

Answer 19

very long reads in the 1000’s of
bases and can deal with repetitive sequences

Question 20

what sort of readings do you achieve with Nanopore/Long-Read sequencing:

Answer 20

very long reads in the 1000’s of
bases and can deal with repetitive sequences

Question 21

downside to Nanopore/Long-Read Sequencing:

Answer 21

The accuracy is not as good as other
methods, so there is significant reliance on consensus alignment

Question 22

consensus of Long-Read/Nanopore Sequencing:

Answer 22

Consensus is where you take multiple
reads that align together and taking what is common across all the reads for the true sequence

Question 23

why apply drug treatment and fund drug development (development)?

Answer 23

Finding new genes involved may help identify new target genes and pathways to develop treatments against

Question 24

why bother with genetic testing?

Answer 24

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

Question 25

genetic testing:

Answer 25

• 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

Question 26

genetic testing:

Answer 26

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

Question 27

genetic testing:

Answer 27

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

Question 28

assessing the results of genetic testing:

Answer 28

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

Question 29

APOE4:

Answer 29

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