DNA Sequencing Techniques | An Overview

Summary

This lecture offers an in-depth overview of DNA sequencing techniques, explaining the process of determining the order of base pairs in DNA molecules. It covers the significance of DNA sequencing in detecting mutations, distinguishing organisms, and identifying human haplotypes. The video explores various sequencing methods, including direct sequencing techniques like Sanger sequencing and chemical sequencing, as well as next-generation sequencing technologies such as ion conductance, reversible dye terminator, sequencing by ligation, and nanopore sequencing. Each method is briefly explained, highlighting its unique approach to DNA analysis.

Takeaways

• 🧬 DNA sequencing is the process of determining the order of base pairs in a DNA molecule, crucial for understanding genetic information.
• 🔎 There are four bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T), which are the building blocks of genetic code.
• 🔍 DNA sequencing helps in detecting mutations, distinguishing organisms, and identifying human haplotypes and polymorphisms.
• 🧪 Direct sequencing methods like chemical sequencing (Maxim Gilbert) and Sanger sequencing allow for direct reading of DNA base sequences.
• 🔬 Chemical sequencing uses chemical agents to cleave DNA at specific base pairs and involves electrophoresis to visualize and read the sequence.
• 🌟 Sanger sequencing is a modification of the DNA replication process using dideoxynucleotides (ddNTPs) to terminate DNA strand growth at specific bases.
• 🔄 Pyrosequencing is an indirect method that detects DNA sequence by monitoring light generation as nucleotides are incorporated into a growing DNA strand.
• 🧵 Bisulfite DNA sequencing is used to detect methylated cytosines, which play a role in gene regulation and chromatin structure.
• 🌐 Next Generation Sequencing (NGS) technologies like ion conductance, reversible dye terminator, and nanopore sequencing allow for rapid, high-throughput DNA sequencing.
• 🧬 NGS techniques are used for large-scale genomic studies and require powerful computing for data analysis and assembly of sequenced libraries.

Q & A

What is DNA sequencing and why is it important?

DNA sequencing is the process of determining the order of base pairs in a DNA molecule. It's important because it can help detect mutations in genes, distinguish between different organisms by analyzing specific gene sequences, and identify human haplotypes and polymorphisms, which provide insights into gene inheritance and function.

What are the four bases found in DNA and how are they abbreviated?

The four bases found in DNA are adenine (A), cytosine (C), guanine (G), and thymine (T).

How does DNA sequencing help in identifying mutations?

DNA sequencing helps identify mutations by determining the exact sequence of bases in a gene. A change in just one base can lead to a change in the gene's function, which can have significant effects on an individual.

What is the purpose of using the 16s rRNA gene in distinguishing microorganisms?

The 16s rRNA gene is used to distinguish microorganisms because it contains universal sequences that are conserved across different species, allowing for the identification of specific differences that can differentiate one microorganism from another.

What are the two main types of direct DNA sequencing techniques mentioned in the script?

The two main types of direct DNA sequencing techniques mentioned are chemical sequencing (Maxim Gilbert sequencing) and Sanger sequencing.

How does chemical sequencing differ from Sanger sequencing?

Chemical sequencing uses chemical agents to cleave DNA at specific base pairs, while Sanger sequencing is a modification of the DNA replication process that uses modified dNTPs (ddNTPs) to create different lengths of DNA fragments.

What is the role of electrophoresis in DNA sequencing?

Electrophoresis is used in DNA sequencing to separate DNA fragments based on their size. The separated fragments are then visualized and the sequence is read from the pattern they form on the gel or in the capillary.

Why is Sanger sequencing still widely used today?

Sanger sequencing is still widely used because it can be adapted for automation, which allows for faster and more efficient sequencing. It uses fluorescent dyes instead of radioactive labels, making it safer and more convenient.

What is pyrosequencing and how does it determine the DNA sequence?

Pyrosequencing is a DNA sequencing method that relies on the generation of light through luminescence whenever nucleotides are added to a growing strand of DNA. It determines the sequence by detecting which nucleotide generates a light signal when added.

What is bisulfite DNA sequencing and why is it used?

Bisulfite DNA sequencing is used to detect methylated cytosines, which play a role in gene expression regulation and chromatin structure. It involves treating DNA with bisulfite to convert cytosines to uracil, leaving methylated cytosines unchanged, and then sequencing to identify the methylated sites.

What are the key features of Next Generation Sequencing (NGS) technologies?

NGS technologies are characterized by their ability to sequence large numbers of templates carrying millions of bases in a short time. They use target libraries, require powerful computers and bioinformatics for data analysis, and include methods like ion conductance sequencing, reversible dye terminator sequencing, sequencing by ligation, and nanopore sequencing.

Outlines

00:00

🧬 DNA Sequencing Techniques Overview

This paragraph introduces the concept of DNA sequencing, which is the process of determining the order of base pairs in a DNA molecule. It explains the significance of knowing the DNA sequence, including detecting mutations, distinguishing between organisms, and identifying human haplotypes. The paragraph outlines two main types of DNA sequencing: direct sequencing, which includes Maxam Gilbert sequencing and Sanger sequencing, and their applications in genetic analysis.

05:02

🔬 Direct Sequencing: Chemical and Sanger Methods

The paragraph delves into the details of direct sequencing methods, starting with chemical sequencing or Maxam Gilbert sequencing. It describes the process of labeling DNA with radioactive isotopes and using chemical agents to cleave DNA at specific bases. The resulting fragments are separated by size through electrophoresis to determine the sequence. The paragraph then contrasts this with Sanger sequencing, which is a modified DNA replication process using dideoxynucleotides (ddNTPs) to terminate DNA strand growth at specific bases, creating fragments that can be sequenced by gel or capillary electrophoresis.

10:03

🌟 Sanger Sequencing and Automation

This section focuses on the Sanger sequencing method, emphasizing its role in automated DNA sequencing. It explains the use of fluorescent dyes instead of radioactive labels for automated reading of sequences. The paragraph outlines two types of automated Sanger sequencing: dye primer and dye terminator sequencing, each with its method of electrophoresis. It also mentions the advantages of capillary electrophoresis in automated sequencing and how the resulting data is interpreted to determine DNA sequences.

15:06

🔍 Indirect Sequencing: Pyrosequencing and Bisulfite Sequencing

The paragraph introduces indirect sequencing methods, starting with pyrosequencing, which detects DNA sequence by monitoring light generation upon nucleotide incorporation. It details the steps involved, from the addition of dNTPs to the detection of light signals and the interpretation of these signals to determine the DNA sequence. The paragraph also covers bisulfite DNA sequencing, a method used to detect methylated cytosines, which involves treating DNA with bisulfite to convert unmethylated cytosines to uracil, followed by sequencing to identify methylation patterns.

20:07

🌐 Next Generation Sequencing (NGS) Technologies

This section discusses Next Generation Sequencing (NGS) technologies, which include ion conductance sequencing, reversible dye terminator sequencing, sequencing by ligation, and nanopore sequencing. It highlights the massive parallel sequencing capabilities of NGS, allowing for the analysis of millions of bases in a short time. The paragraph describes each NGS method, explaining how they work, the type of libraries they use, and the equipment and bioinformatics required for data processing and analysis.

25:09

🧪 Sequencing by Ligation and Nanopore Sequencing

The final paragraph focuses on sequencing by ligation, which uses oligomers that hybridize and ligate to the DNA template, allowing for the detection of two bases at a time. It also covers nanopore sequencing, a unique method that sequences a single long DNA molecule by identifying each nucleotide as it passes through a protein pore, causing a disruption in the current. The paragraph provides insights into the technical aspects of these advanced sequencing techniques and their applications in genomic research.

Keywords

💡DNA Sequencing

DNA sequencing refers to the process of determining the exact order of nucleotides within a DNA molecule. It is central to the video's theme as it sets the stage for discussing various methods of sequencing. The video mentions that knowing the sequence can help detect mutations, distinguish organisms, and identify human haplotypes, emphasizing its importance in genetic research and medical diagnostics.

💡Base Pairs

Base pairs are the building blocks of DNA, consisting of adenine (A), cytosine (C), guanine (G), and thymine (T). These pairs are crucial for DNA sequencing as their sequence determines genetic information. The video uses the example of the 16s rRNA gene in E. coli to illustrate how around 1500 base pairs are sequenced to understand genetic makeup.

💡Mutations

Mutations are changes in the DNA sequence, which can potentially alter gene function. The video explains that DNA sequencing can detect these mutations, which might have significant effects on an individual's health. This keyword is vital for understanding the practical applications of sequencing in identifying genetic disorders or variations.

💡Direct Sequencing

Direct sequencing is a method that identifies genetic sequences by directly reading the base sequences, often involving electrophoresis. The video describes two types of direct sequencing: chemical sequencing and Sanger sequencing. This concept is key to understanding traditional sequencing techniques.

💡Chemical Sequencing

Chemical sequencing, also known as Maxam-Gilbert sequencing, uses chemical agents to cleave DNA at specific base pairs. The video explains how this method involves labeling DNA with radioactive materials and then using chemicals like dimethyl sulfate to break DNA strands, which are then sequenced through electrophoresis. It's an older technique, illustrating the evolution of sequencing methods.

💡Sanger Sequencing

Sanger sequencing, also known as dideoxy chain termination sequencing, is a widely-used method that modifies the DNA replication process. The video describes how it uses ddNTPs to create fragments of different lengths, which are then sequenced through electrophoresis. Sanger sequencing is highlighted for its adaptability to automation, making it a significant technique in modern genomics.

💡Pyrosequencing

Pyrosequencing is an indirect sequencing method that detects DNA sequences by monitoring light signals generated during nucleotide incorporation. The video explains how it uses enzymes and substrates to convert the release of pyrophosphate into light, which is then detected to determine the sequence. This method is noted for not requiring a sequencing ladder, streamlining the sequencing process.

💡Bisulfite DNA Sequencing

Bisulfite DNA sequencing is used to detect methylated cytosines, which play a role in gene regulation. The video describes how it treats DNA with bisulfite, converting unmethylated cytosines to uracil while leaving methylated cytosines unchanged. This keyword is important for understanding epigenetic studies and their impact on gene expression.

💡Next Generation Sequencing (NGS)

NGS encompasses various high-throughput sequencing techniques that can sequence millions of DNA fragments simultaneously. The video mentions methods like ion conductance sequencing and reversible dye terminator sequencing. NGS is highlighted for its ability to analyze entire genomes or gene panels, marking a significant advancement in sequencing technology.

💡Ion Conductance Sequencing

Ion conductance sequencing is an NGS method that detects DNA sequences by measuring changes in pH when nucleotides are incorporated. The video describes how it uses microparticles and a gene chip to amplify and sequence DNA. This method is noted for its high throughput and accuracy, making it suitable for large-scale genomic studies.

💡Sequencing by Ligation

Sequencing by ligation is an NGS technique that uses fluorescently labeled oligomers to sequence DNA. The video explains how it detects two bases at a time by ligating complementary oligomers to the template DNA. This method is unique for its ability to provide high accuracy and is an example of the innovative approaches in NGS.

Highlights

Introduction to DNA sequencing, the process of determining the order of base pairs in DNA.

Explanation of the four DNA bases: adenine (A), cytosine (C), guanine (G), and thymine (T).

Overview of the 16s rRNA gene in E. coli and its 1500 base pairs.

Applications of DNA sequencing include mutation detection and organism identification.

Direct sequencing methods, such as chemical sequencing and Sanger sequencing, are introduced.

Description of chemical sequencing, including the use of radioactive labels and chemical agents.

Process of Sanger sequencing, involving the use of ddNTPs and electrophoresis.

Advantages of Sanger sequencing, including its adaptability for automation.

Introduction to indirect sequencing methods like pyrosequencing, which relies on luminescence.

Bisulfite DNA sequencing for detecting methylated cytosines and its role in gene regulation.

Overview of Next Generation Sequencing (NGS) technologies, including their high-throughput capabilities.

Ion conductance sequencing and its detection of hydrogen ions released during DNA synthesis.

Reversible dye terminator sequencing and the formation of DNA colonies on a flow cell.

Sequencing by ligation, which detects two bases at a time using fluorescently labeled oligomers.

Nanopore sequencing, a single-molecule sequencing method that identifies nucleotides by current disruption.

Practical applications and the importance of bioinformatics in reassembling sequenced libraries.

Transcripts

hi everyone

in this lecture we will have an overview

of the different dna sequencing

techniques

now what is dna sequencing this is the

process of determining the sequence or

order of base pairs in a given molecule

of dna

as a recap there are four bases

in your dna and these are adenine

abbreviated by a

cytosine which is abbreviated by c

guanine which is abbreviated by g and

thymine which is abbreviated by t

in this example you can see the sequence

of the 16s rrna gene in e coli and you

can see there are about 1500 base pairs

and we know this sequence because of dna

sequencing

so why do we need to know the sequence

of dna or certain genes

here are just some applications first

it can help us detect mutations in

specific genes of interest

in this example you can see that this

gene is composed of around 1 500 bases

but just change to one of these bases

can lead to more function of the gene

and even detrimental effects to an

individual

next

knowing the sequence of a specific gene

can help us distinguish one organism

from another this is especially useful

in identifying microorganisms which can

be differentiated from each other by

looking at specific differences

in universal genes like in this example

the 16s rna gene

it can also be used to help us identify

human haplotypes and designate

polymorphisms

this gives us information on how genes

are inherited and how these variations

might affect a gene's function

now let's go into

the different types of dna sequencing

techniques the first

grouping of dna sequencing is called

direct sequencing

and here there are two examples the

maxim gilbert sequencing and sanger

sequencing

so what is direct sequencing

this is the most definitive molecular

method to identify genetic lesions and

to know the sequence of dna

here the base sequences are read

directly and usually direct sequencing

methods involve some sort of

electrophoresis in which you can

visualize bands that correspond to

whatever base is in the sequence

this is especially useful in detecting

small variations in a specific sequence

the first type of sequencing we have is

called chemical sequencing or maxim

gilbert sequencing

this type of technique requires single

single-stranded dna or double-stranded

dna with a radioactive label at one end

usually this is the 5-prime end

this type of sequencing is called

chemical sequencing because it uses

chemical agents to cleave dna

at specific base pairs

different fragments are then separated

based on size to identify the sequence

the first step in chemical sequencing is

the addition of a radioactive label

the label being used is a radioactive

phosphorus and usually this is

conjugated to atp

this is added to the five prime end of a

dna fragment using your polynucleotide

kinase enzyme

if you wanted to add

this radioactive phosphorus to the three

prime end the usual method is by using

terminal transferase plus alkaline

hydrolysis to remove excess adenylic

acid residues

in this figure you can see that you take

our template dna and after this step

they will now be labeled with a

radioactive label now the radioactive

label here is very important in order

for us to visualize the dna fragments

later on

the next step is to use chemical agents

to break apart the fragment

so our template dna which has already

been labeled is divided into four

each aliquot is treated with a different

chemical and there are four being used

first we have dimethyl sulfate

abbreviated by dms

formic acid which is abbreviated by fa

hydrazine which is abbreviated by h and

a combination of hydrazine and a salt

abbreviated here with h plus s

in this figure you can see our four

tubes each containing

one of our different chemical agents

so we add our template dna to each tube

and incubate it for a period of time

then a strong reducing agent such as 10

percent pipiridine is added which breaks

the strand at specific nucleotides what

you are left with

is different fragments of different

sizes each ending in a specific

nucleotide depending on which

compound or which chemical it was

incubated with

in this table we can see the different

chemical agents are base modifiers being

used in chemical sequencing

so these base modifiers break apart

your dna fragment at specific

nucleotides and it does these using

these different actions

we can also see here the time it takes

or the incubation time for each of these

base modifiers to completely break apart

your template dna

so first we have dimethyl sulfate or dms

and this breaks the dna fragment

whenever it encounters a guanine

formic acid breaks apart the dna

fragment whenever it encounters a

guanine and an adenine then we have

hydrazine which breaks apart the chain

whenever it encounters a thymine and a

cytosine and we have hydrazine salt

which

breaks apart the chain whenever it

encounters a cytosine

the last step in chemical sequencing is

the separation of different fragments

and the reading of the sequence

in the previous step we are left with

different fragments and depending on the

chemical agents being used we are sure

that these fragments end in a certain

nucleotide

now these fragments are loaded into a

gel and separated based on their size

using electrophoresis the different

fragments are separated by size on a

denaturing polyacrylamide gel by

electrophoresis

the radioactive label which was added in

step one is used to visualize the bands

either using autoradiography or by

exposing the gel to an x-ray film

the sequence is read from the bottom to

the top and this is from the five prime

end all the way to the three prime end

in this figure you can see an example of

a gel being used in chemical sequencing

so at the bottom you will find the

shortest fragments and this is closer to

the five prime end this is a recap we

added the primer in the five prime end

okay and in the top here you have much

longer fragments

now the lane in which the band appears

is used to identify the nucleotide so

for example if you have a band in the g

and the g plus a lane then the

nucleotide in that sequence is a guanine

if you have a band both in the c plus t

lane and the c lane then the nucleotide

is a cytosine

if you have a band only in the c plus t

lane the nucleotide is a thymine and if

you have a band only in the g plus a

lane then the nucleotide is an adenine

so reading this gel from the bottom this

is the five prime end you can see that

the first band we can see is found in

the g plus a lane only this indicates

that the first nucleotide in the

sequence

is an a

then the following

bands are found in the c plus t

and the c lane and we have two bands so

this tells us the next basis in the

sequence is a c and a c

and we just go through this gel until

you reach the top so in the top you can

see here that

there is a band in the c plus t lane

only and this tells us that

our

next base in the sequence is a t

chemical sequencing also comes with some

disadvantages it can only be used for

sequencing short lengths of dna

and it uses hazardous materials like

your hydrazine and your pipiridine and

this means that you need special

equipment and specially trained

individuals in order to perform our

chemical sequencing

the next type of direct sequencing is

called your die deoxy chain termination

or sanger sequencing

this is a modification of the dna

replication process

it uses single stranded template dna and

a single stranded primer

in this type of sequencing a modified

dntp called

dioxynucleotide or ddntp is used to

create different lengths of dna

fragments

and the fragments are run through a gel

or capillary electrophoresis in order to

tell the sequence of the dna fragment

the first step in your sanger sequencing

is the addition of the different

reactants here are the different

reactants being used first we have your

template dna this will be your pcr

product then you have your primer so the

primer binds to the three prime end of

the template and it creates copies of

the template from the five prime end to

the three prime end

sometimes the primer can be conjugated

with a radioactive phosphorus or a

fluorescent dye as a label

other reactants that we use

are the dntps just as a recap dntp

stands for

deoxyribonucleoside triphosphate but we

just call them dntps for short

these are the building blocks of our dna

whenever we do

amplification

we have four dntps one each for adenine

guanine cytosine and thymine

then in sanger sequencing we have these

ddntps

which are like your regular dntps but

they lack the hydroxyl group found on

the third ribose carbon

of the deoxynucleotide so here we have

our regular dntp and our d dntp

and here you have

the missing hydroxyl group

whenever our ddntps are added to a

growing sequence of dna

this terminates the replication process

of course you also need the other

components for dna replication like your

polymerase and other

substrates that it may use

the next step in sanger sequencing is

the dna replication so the reactions

occur in four different tubes and each

tube has the four dntps and one specific

ddntp at a lower concentration

whenever a ddntp is added to a growing

dna strand the replication stops and

this results in different length strands

each ending with a specific dntp

in this figure you can see our four

different tubes

again each tube has

a specific ddntp and all tubes have the

four dntps which are essential building

blocks

for dna replication

it should also be noted that the ratio

between ddntps and dntp in each tube

must be optimized if there is too much

dntp then you will be left with very

very short fragments and if there is not

enough ddndp you will end up with very

long fragments

so you need an optimized ratio of the

two in order to get a variety of

different fragments which will allow you

to easily sequence your dna fragment

the last step in our sanger sequencing

is the separation of fragments and the

actual sequencing

to do this a sequencing ladder is first

created here you can see an example

of a sequencing ladder

this is still a polyacrylamide gel

electrophoresis similar to our chemical

sequencing

each reaction is drawn on a different

lane so we have four lanes here

and since we know that each reaction

contains a specific ddntp we can

associate bands in each lane with a

specific nucleotide base so we have one

for a c b and g

the sequence is read based on the

position and the lane of the band

so the lane in which the band can be

found will tell us the base identity

and

the position of the band or the

migration length will tell us the

nucleotide sequence

so shorter fragments have a faster

migration

bands that migrate farther tell us that

the nucleotide is closer to the five

prime end

where we have our primer and

the larger bands

will migrate less which tells us that

these nucleotides are much farther away

from our primer

so when we read this gel for example you

read it from the bottom to the top that

is the five prime end to the three prime

n

the first band we see here is found in

the lane for a

so this means that the first nucleotide

in our sequence is a

here you have a g

the next in the sequence is a c

followed by another g and by a t so you

just read all of these bands until you

get to the top band which is your g

sanger sequencing is still being widely

used today mainly because it can be

adapted for automation

in an automated sequencing instead of

using radioactive dyes we use different

fluorescent dyes corresponding to hd

ddntp to allow for automated reading of

the sequencing ladder

there are two types of automated sanger

sequencing

first we have die primer sequencing

in this sequencing the dye is attached

to our primer as the name suggests and

we have diet terminator sequencing here

the dye is being attached or conjugated

through the dntp

automated science sequencing can also

employ two different types of

electrophoresis first you have the more

traditional gel electrophoresis which

can either use dye primer sequencing or

die terminator sequencing

and the more popular capillary

electrophoresis which can only use diet

terminator sequencing

capillary electrophoresis is more widely

used because it allows for all four

sequencing reactions to be performed

in the same tube

each band has its own corresponding

fluorescent color and this is caused by

the ddntp which terminates each fragment

after the machine

reads the fluorescence it comes up with

this electro ferrogram

here you can see a variety of peaks and

these indicate the fluorescence which

was detected by your machine

each peak is a different color of

fluorescence and this corresponds to a

specific nucleotide for example this

first peak

is corresponding to our g nucleotide a

and then followed by two peaks which

correspond to two t's and this continues

until the entire sequence is red

now depending on the reagents and the

gels being used the number of bases per

sequence read averages from 300 to 500

bases per read

next we'll talk about the indirect

methods for sequencing dna the first is

pyrosequencing

unlike your direct sequencing methods

which

directly show us the different bands

which correspond to the different

nucleotides pyrosequencing relies on the

generation of light through luminescence

whenever nucleotides are added to a

growing strand of dna

this is designed to determine a dna

sequence without having to make a

sequencing ladder here you can see an

example of a machine by illumina which

is one of the most common machines being

used for pyrosequencing in fact one of

the common names for pyrosequencing is

also called illumina sequencing

here you can see the different

components in a sequencing reaction

these include our single stranded dna

templates

our sequencing primer and some enzymes

which include sulfurylase and luciferase

and their substrates adenosine 5

phosphosulfate or aps and luciferin

the first step in pyrosequencing is the

addition of individual dntps we have our

template dna which is usually our pcr

product and this is immobilized in

individual flow cells or wells

then these wells are flooded with only

one type of dntp at a time

if the added dntp is complementary to

the template it is added to the strand

and pyrophosphate is released through

this

reaction

so this is your pyrophosphate

the next step in pyrosequencing is

luminescence and detection

previously we were left with our

pyrophosphate now sulfurolase combines

pyrophosphate and aps into atp

next this adp is used by luciferase to

convert luciferin into oxyluciferin and

in this reaction also this produces

light

now the light here is detected by our

luminometer

so our nucleotides are only added one at

a time to each well and the sequence is

determined by which the four nucleotides

generates a light signal so for example

if the light signal was detected when we

added

a guanine dntp then we can tell that the

next base in the sequence is guanine if

the light was seen when we added our

cytosine dntp then we can tell that the

next base in the sequence is your

cytosine

the last step in pyrosequencing is

resetting the system

here aparase is used to remove excess

free dntp and datp so that another dntp

can be added and here you can see the

reactions

that api race does in order to remove

these excess reactants

pyrosequencing machines produce

pyrograms this is a graphical

representation of what the machine was

able to observe during the reaction

this consists of peaks of luminescence

associated with the addition of

complementary nucleotide here at the

bottom you can see each instance where a

nucleotide was added and whenever a

luminescence was observed you can see a

peak so for example here when the g and

the c nucleotides were added the machine

was able to observe some luminescence

corresponding to this peak right here

so the machine will add g and c to the

nucleotide sequence however when we

added the t nucleotide no luminescence

was observed so

no peak is formed and the machine

foregoes this nucleotide in the sequence

or it skips it

repeated nucleotides show larger

luminescence peak height for example

here when the g nucleotide was added

a larger luminescence was observed and

this indicates that

at this

part of the sequence there are two g's

and the same can also be seen here with

two c's

next we have bisulfite dna sequencing

this type of dna sequencing is also

known as methylation-specific sequencing

and it's used to detect methylated

cytosines these methylated cytosines are

an important player in the regulation of

gene expression and chromatin structure

so sometimes although our dna may not

have any mutations the presence of these

different cytosines might still cause

some genes to be downregulated or even

inactivated

the first step in bisulfite dna

sequencing is bisulfite conversion

here dna is fragmented and purified then

the different fragments are denatured

with heat at approximately 97 degrees

for five minutes and exposed to a

bisulfite solution this solution is

composed of sodium bisulfite sodium

hydroxide and hydrokenone for 16 to 20

hours

what this reaction does it converts

regular cytosines into uracil however if

those cytosines are already methylated

then this reaction will leave them

unchanged

the next step is sequencing the treated

fragments are sequenced using sanger

sequencing or pyrosequencing and the

degree of methylation is determined by

comparing our bisulfite treated setups

with our untreated dna fragments

here you can see an example of a

pyrogram showing you the different

converted cytosines they are

denoted here by the t

which stands for your uracil

and you also have your unchanged

cytosines

which are here seen as c with an

underscore

now we compare these with the untreated

dna fragments in order to find the

degree of methylation

next we will talk about next generation

sequencing or ngs

these include methods like your ion

conductance sequencing reversible die

terminator sequencing sequencing by

ligation and nanopore sequencing

now what is next generation sequencing

they are also known as massive parallel

sequencing techniques and they were

designed in recent years to sequence

large numbers of templates carrying

millions of bases in a short time period

they are usually used for genomic or

gene panel studies and they have these

common characteristics first they use

target libraries which are a collection

of dna fragments to be sequenced

usually these fragments may be labeled

or indexed

they also need to use very powerful

computers and bioinformatics to

reassemble the sequenced libraries and

give us information about the gene or

genome of interest

the first ngs technique we'll be

discussing is called ion conductance

sequencing this technique uses indexed

libraries otherwise known as gene panels

and they are amplified using primers

immobilized on micro particles so here

you can see a microtube containing a

variety of microparticles or beads

and our template dna attaches to these

beads and are amplified using epcr or

emulsion pcr

next

these beads carrying the amplicons or

our sequence templates are placed on a

solid surface gene chip

next nucleotides are added in a

predetermined order or one at a time

and when a complementary dntp is added a

hydrogen ion is released and you can see

that in this figure right here so aside

from pyrophosphate which was discussed

in our pyrosequencing hydrogen ions are

also released whenever a

new nucleotide is added to a growing

sequence

the hydrogen ion will lower the ph of

the reaction by a specific amount and

this is recorded by a sequencer so

whenever a change in ph is detected

whatever dntp was added

to the plate is recorded as what is next

in the sequence

the next ngs technique we'll be

discussing is called reversible die

terminator sequencing

here amplified fragments are hybridized

to immobilize primers on a solid surface

called a flow cell

the fragments hybridized to the

immobilized primers and are amplified

using a special pcr called branch pcr

and this forms clusters of products

called colonies

so this is what a

flow cell looks like and a microscopic

look at the flow cell will show you

these different primers so we have a

primer for reverse and a forward primer

so through our branch pcr these clusters

of

dna are formed

called our colonies

so once these different colonies are

created they are sequenced in place by

the sequential addition of fluorescently

labeled nucleotides

so here you can see

an example of our different colonies and

we add a variety of nucleotides to them

these nucleotides have a specific

fluorescent label so whenever we detect

the light coming from a specific

nucleotide that nucleotide is then added

to the sequence of this colony

next we'll talk about sequencing by

ligation

unlike the other techniques which use

individual dntps sequencing by ligation

uses short fluorescently labeled

oligomers that hybridize in short

increments if they are complementary to

the dna template when we say oligomers

these refer to short chains of nucleic

acid

template dna anchored to a glass slide

is flooded with a fluorescent labeled

oligonucleotide

and if the oligonucleotide is

complementary to the template it is

ligated by dna ligase

using this method two bases are detected

at a time

oligonucleotide is cleaved followed by

the next round of ligation each time two

new nucleotides are detected

so in this figure you can see a

graphical representation of our

sequencing by ligation here you have our

different oligomers and each type of

oligomer has a specific fluorescent

probe attached

once the oligomer attaches to the

template strand ligation is done

followed by

detection

then

cleavage of the unused oligomer so that

a new oligomer can bind to the template

lastly we will talk about nanopore

sequencing

unlike other methods nanopore sequencing

is unique because it does not require

fragmentation and amplification of the

template dna instead it uses one long

double-stranded dna molecule up to 1

megabase pairs long this is equivalent

to 1 million base pairs and this is

drawn through a protein pore

each nucleotide is identified by a

disruption in the current as it passes

through the pore so here we have an

example of our different protein pores

and as the dna passes through this pore

each

base pair causes a change in the current

which is then detected by your sequencer

here you can see the different changes

in the current by our different

nucleotide bases

if you wanted to learn more about the

things we just discussed

please check out this

reference

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