Name:- Muhammad Jahanzaib

Roll no. 1006

Assignment:- Introduction to Omics
Submitted to:- Dr. Sohail Raza
Second Generation Sequencing:-

Second generation sequencing also known as Next-generation sequencing (NGS) is a massively

parallel sequencing technology that offers ultra-high throughput, scalability, and speed. The
technology is used to determine the order of nucleotides in entire genomes or targeted regions of
DNA or RNA. NGS has revolutionized the biological sciences, allowing labs to perform a wide
variety of applications and study biological systems at a level never before possible.  

Today's complex genomics questions demand a depth of information beyond the capacity of
traditional DNA sequencing technologies. NGS has filled that gap and become an everyday tool to
address these questions.

Steps in Next (2nd) Generation Sequencing:-

The 4 steps of next generation sequencing (NGS) include nucleic acid isolation, library
preparation, clonal amplification and sequencing, and data analysis.

Step 1- Nucleic Acid Extraction and Isolation

Nucleic acid extraction and isolation is a vital first step in next generation sequencing. This is
regardless of whether you are sequencing total RNA, genomic DNA, or various RNA types. The
extraction method that’s used will depend on the starting material. It is crucial to choose an
extraction protocol that’s optimized to yield the maximum amount and highest purity of nucleic
acid from the respective sample type. The yield, quality and integrity of isolated nucleic acids are
critical for successful sequencing and must be assessed before proceeding to the next step.

Step 2- Library Preparation

Library preparation involves preparing DNA or RNA samples so they can be processed and read
by sequencers. This is done by fragmenting the samples to yield a pool of appropriately sized
targets, then adding specialized adapters at both ends, which will later interact with the NGS
platform. These prepared, ready-to-sequence samples are called “libraries”. A library represents
a collection of molecules that can be sequenced. The exact library preparation procedure may
differ depending on the reagents and methods used. Regardless of the procedure used, the final
prepared NGS libraries must contain DNA fragments of desired lengths with adapters at both
ends.

Step 3- Clonal Amplification and Sequencing

Clonal amplification involves amplifying DNA fragments to be sequenced by binding to ion

surfaces, beads or flow cells. This helps develop strong fluorescent signals that can be detected
by the sequencers. 

Sequencing by synthesis (SBS) is the next step after clonal amplification. In this step the library
is loaded onto the sequencer, which then ‘reads’ or detects the nucleotides one by one. 
Step 4 -Data Analysis Using Bioinformatics

This final step involves three stages – processing, analysis, and interpretation of the raw
sequencing data generated. A variety of bioinformatics tools are used to process, analyze, and
interpret the raw sequencing data and convert it into meaningful information. The exact tools
used as well as how the data is processed and analyzed depends on the applications and goals of
the NGS assay.

Applications of NGS:-

Next-generation sequencing technology has fundamentally changed the kinds of questions scientists
can ask and answer. Innovative sample preparation and data analysis options enable a broad range
of applications. For example, NGS allows labs to:

 Rapidly sequence whole genomes

 Deeply sequence target regions
 Utilize RNA sequencing (RNA-Seq) to discover novel RNA variants and splice sites, or
quantify mRNAs for gene expression analysis
 Analyze epigenetic factors such as genome-wide DNA methylation and DNA-protein
interactions
 Sequence cancer samples to study rare somatic variants, tumor subclones, and more
 Study the human microbiome
 Identify novel pathogens
Short Read Sequencing:-

To sequence a large genome like human DNA using NGS, the DNA has to be fragmented and
amplified in clones of between 75 base pairs and 400 base pairs, hence the term 'short-read
sequencing' (SRS). Computer programs are then used to assemble the random clones into a
contiguous sequence.

Sequencing by Synthesis or Ligation:-

Short-read technologies carry out sequencing by synthesis or ligation. Each strategy uses DNA
polymerase or ligase enzymes, respectively, to extend numerous DNA strands in parallel.
Nucleotides can either be provided one at a time, or they can be modified with identifying tags.

Short-read sequencing technologies can be further categorized as either single molecule-based,

involving the sequencing of a single molecule, or ensemble-based, which is the sequencing of
multiple identical copies of a DNA molecule that have usually been amplified together on
isolated beads.
Furthermore, these methods could be real-time or synchronous controlled. Real-time short-read
sequencing consists of a free-running DNA polymerase that catalyzes all possible nucleotides.
Therefore, this method requires the identification of the newly sequenced nucleotides as they are
being incorporated, without interrupting the synthesis process. This can be done using optical or
physical techniques, which reveal tagged nucleotides that are either free or bound. The bound
nucleotides are assumed to be involved in DNA synthesis. Synchronous-controlled approaches
use information to facilitate the identification process in an interrupted fashion, which can be
achieved by adding a single type of nucleotide at once or using nucleotide reversible
terminators..

Here are some examples of short-read sequencing technologies:

 Illumina – Single-stranded DNA-binding proteins are used for amplification, followed

by the addition of fluorescent-labelled deoxynucleoside triphosphates to bridge the
amplified DNA template.
 454 pyrosequencing – Clonal amplification is done by emulsion PCR, generating
microbead-bound DNA clones. Emulsion PCR allows the amplification of DNA in
physically separated water-in-oil droplets, avoiding unwanted reactions between similar
DNA sequences. DNA polymerase is added and the nucleotides are washed.
Deoxynucleoside triphosphate incorporation is then monitored by pyrophosphate release.
 Ion Torrent – Clonal amplification is done by emulsion PCR, generating microbead-
bound DNA clones. DNA polymerase is added, and nucleotides are washed.
Deoxynucleoside triphosphate incorporation is then monitored by a pH sensor as protons
are released.
 SOLiD – Clonal amplification is done by emulsion PCR. The DNA template, which is
bound to microbeads, is flanked by adapters and is hybridized to the growing
complementary strand by DNA ligase.
 cPAL – The anchor sequence and probes hybridize to the DNA template in a series of
ligation reactions taking place on a nanoball.