Next generation sequencing by Muhammad Abbas

1. Next Generation Sequence (NGS)
Muhammad Abbas
Next Generation Sequence | M. Abbas | 15-03-2018 1

2. Objectives
Introduction
Definition
Procedure
NGS workflow
Purpose
Software for data analysis
Application
Summary
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3. Introduction
Since first introduced to the market in 2005, next-generation
sequencing technologies have had a tremendous impact on genomic
research.
The next-generation technologies have been used for
standard sequencing applications, such as genome sequencing and
resequencing, and for novel applications previously unexplored by
Sanger sequencing.
Ref: O. Morozova, M.A. Marra / Genomics 92 (2008) 255–264
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4. Cont.…
Currently molecular diagnostics of human pathogens provide limited
information that is often not sufficient for outbreak and exactly
investigation.
Next generation sequencing (NGS) determines the DNA
sequence of a complete genome in a single sequence run, and from
these data, information on resistance and virulence, as well as
information for typing is obtained, useful for outbreak investigation.
Ref: R.H. Deurenberg et al. / Journal of Biotechnology 243 (2017) 16–24
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5. Cont.…
Very recently, the Sanger method has been partially supplanted by
several “next-generation” sequencing technologies that offer dramatic
increases in cost-effective, and time saving sequencing in the genomic
world.
The next-generation technologies commercially available today
include the 454 GS20 pyrosequencing based instrument (Roche Applied
Science), the Solexa 1G analyzer (Illumina, Inc.), the SOLiD instrument
from Applied Biosystems, and the Heliscope from Helicos.
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6. Definition of Next Generation Sequence
 Determining the order of bases in a proper sequence i-e A,G,C,T
• “Massively Parallel Sequencing: One can generate hundreds of millions of
short sequences (up to 250bp) in a single run in a short period of time .”
Modern high-throughput DNA sequencing technologies.
parallel, rapid .
Decreasing price, time, workflow complexity, error rate.
Sequence comparison with reference genome.
Wide range of applications.
Ref: Michael Metzker (2010) Nature Reviews Genetics 11:31 Quail et al (2012) BMC Genomics Jul 24;13:341
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7. NGS (Next Generation Sequence) allows sequencing of the whole genome
of numerous pathogens in one sequence run, either from bacterial
isolates of (different) patients, or from multiple species present in
patient material from one individual.
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9. Procedure
Although different machines have been developed with various
differing technical details, they all share some common features which
are outlined below.
1. Sample Preparation/ Library preparation
All Next Generation Sequencing platforms require a library
obtained either by amplification or ligation with custom adapter
sequences. These adapter sequences allow for library hybridization to
the sequencing chips and provide a universal priming site for
sequencing primers.
Ref: DNA Sequencing with chain-terminating inhibitors. Snager, F, Nicklen, S and Coulson, AR. s.l. : Proc
NatI Acad Sci USA, Vol. 74, pp. 5463-5467
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11. 2. Sequencing machines
Each library fragment is amplified on a solid surface (either beads
or a flat silicon derived surface) with covalently attached DNA linkers
that hybridize the library adapters.
This amplification creates clusters of DNA, each originating from a
single library fragment; each cluster will act as an individual sequencing
reaction
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12. 3. Data Analysis
Each machine provides the raw data at the end of the
sequencing run. This raw data is a collection of DNA sequences that
were generated at each cluster. This data could be further sequencnitlly
aligned using a reference genome or Reference DNA analyzed to
provide more meaningful results.
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13. NGS workflow
• Sample
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Library preparation Sequencing Bioinformatics

14. Actual Sequencing Platforms
• Roche/454 (GS FLX+/GS Junior)
• Illumina Genome Analyzer (HiSeq/MiSeq/NextSeq)
• Life Technologies (3500 Genetic Analyzer,
Ion Torrent Proton/PGM)
• Pacific Biosciences (PACBIO RSII)
• Applied Biosystems (SOLiD, 3730xl DNA Analyzer )
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15. Purpose of Sequencing
Deciphering “code of life”
Detecting mutations
Typing microorganisms
Identifying human haplotype (a set of genetic determinants located on a single chromosome.)
Designating polymorphisms
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16. Software for data analysis
Huge amount of data to handle
Many tools are command-based tools
Make use of a bioinformatics server.
After sequencing, the sequenced fragments (reads) can be assembled
(genome assembly). Hereby, the reads are aligned against each other
without the use of a reference organism.
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17. Applications
Mutation discovery
Map the genome of microorganisms
 Transcriptome Analysis
Sequencing clinical isolates in strain-to-reference
mechanisms.
 Enabling Metagenomics (Metagenomics is the study of genetic material recovered directly
from environmental samples. The broad field may also be referred to as environmental genomics)
 Defining DNA-Protein interactions
Discovering non-coding RNAs
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18. DNA sequencing may be used to determine the sequence of individual
genes, larger genetic regions, full chromosomes or entire genomes.
• Depending on the methods used, sequencing may provide the order
of nucleotides in DNA or RNA isolated from cells of animals, plants,
bacteria or virtually any other source of genetic information.
Ref: SOLiD. Applied Biosystems. [Online] 2015. [Cited: 6 2, 2015.]
http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-
sequencing.html.
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19. • The resulting sequences may be used by researchers in molecular
biology or genetics to further scientific progress or may be used by
medical personnel to make treatment decisions or aid in genetic
counselling.
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20. Summary of Next generation sequencing
Next generation sequencing enables sequencing billions of DNA
fragments simultaneously.
Huge amount of sequence data in a short time.
Highly applicable in many areas of biology and Medicine.
 Needs bioinformatics to handle and analyze the produced data.
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22. Thank You
Presented & Prepared by:
Muhammad Abbas
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