DNA

SEQUENCING
 WHAT IS DNA SEQUENCING?

• SEQUENCING IS THE OPERATION OF DETERMINING THE PRECISE

ORDER OF NUCLEOTIDES OF A GIVEN DNA MOLECULE. IT IS USED
TO DETERMINE THE ORDER OF THE FOUR BASES ADENINE (A),
GUANINE (G), CYTOSINE (C) AND THYMINE (T), IN A STRAND OF
DNA.
• DNA SEQUENCING IS USED TO DETERMINE THE SEQUENCE OF
INDIVIDUAL GENES, FULL CHROMOSOMES OR ENTIRE GENOMES
OF AN ORGANISM. DNA SEQUENCING HAS ALSO BECOME THE
MOST EFFICIENT WAY TO SEQUENCE RNA OR PROTEINS.
• THE SEQUENCE TELLS SCIENTISTS THE KIND OF GENETIC INFORMATION THAT IS CARRIED IN A
PARTICULAR DNA SEGMENT. FOR EXAMPLE, SCIENTISTS CAN USE SEQUENCE INFORMATION TO
DETERMINE WHICH STRETCHES OF DNA CONTAIN GENES AND WHICH STRETCHES CARRY
REGULATORY INSTRUCTIONS.
• IN ADDITION, AND IMPORTANTLY, SEQUENCE DATA CAN HIGHLIGHT CHANGES IN A GENE
THAT MAY CAUSE DISEASE.
• THE HUMAN GENOME CONTAINS ABOUT 3 BILLION BASE PAIRS THAT SPELL OUT THE
INSTRUCTIONS FOR MAKING AND MAINTAINING A HUMAN BEING.
 HISTORY OF DNA
SEQUENCING
THE WORK CARRIED OUT BY A BRITISH BIOCHEMIST
NAMED FREDERICK SANGER, LAID THE FOUNDATION
FOR SEQUENCING PROTEINS. IN 1955, SANGER HAD
COMPLETED THE SEQUENCE OF ALL THE AMINO ACIDS IN
INSULIN. HIS WORK PROVIDED EVIDENCE THAT PROTEINS
CONSISTED OF CHEMICAL ENTITIES WITH A SPECIFIC
PATTERN, RATHER THAN A MIXTURE OF SUBSTANCES.
• LATER, A METHOD NAMED AS SANGER SEQUENCING WAS DEVELOPED BY FREDERICK SANGER
AND HIS COLLEAGUES IN 1977, WHERE DNA COULD BE SEQUENCED BY GENERATING
FRAGMENTS. IT WAS THE MOST WIDELY USED SEQUENCING METHOD FOR APPROXIMATELY 40
YEARS.
PROGRESSION OF SEQUENCING TECHNOLOGY

• GENOMICS IS A RELATIVELY NEW FIELD; IN FACT, THE FIRST DNA SEQUENCES WERE ONLY
OBTAINED LESS THAN HALF A CENTURY AGO IN THE EARLY 1970S. AT THIS TIME, RESEARCHERS
RELIED ON TWO-DIMENSIONAL CHROMATOGRAPHY TECHNIQUES TO SEQUENCE THE DNA, WHICH
WAS VERY TIME-CONSUMING.
• IN RECENT DECADES THERE HAVE BEEN SIGNIFICANT ADVANCEMENTS IN THE TECHNOLOGY
AVAILABLE FOR DNA SEQUENCING. FLUORESCENCE-BASED SEQUENCING METHODS WERE
INTRODUCED TO INCREASE BOTH THE EASE AND SPEED OF RESEARCH.
• THE IMPROVEMENT IN TECHNOLOGY HAS CORRESPONDED TO A SIGNIFICANT INCREASE IN THE
RATE OF SEQUENCING AND ACCELERATED GROWTH IN RELATED RESEARCH.
• THIS HAS ENABLED THE ENTIRE HUMAN GENOME TO BE SEQUENCED, IN ADDITION TO MANY OTHER
SPECIES OF PLANTS AND ANIMALS
 PRESENT AND FUTURE SEQUENCING
TECHNOLOGY
• AT THIS TIME, THERE ARE VARIOUS METHODS AND TECHNOLOGIES THAT CAN HELP IN THE PROCESS
TO SEQUENCE THE DNA. SOME LABORATORIES ARE NOW ABLE TO SEQUENCE AN EXCESS OF
100,000 BILLION NUCLEOTIDE BASES EACH YEAR.
• THE PROCESS HAS ALSO REDUCED SIGNIFICANTLY IN COST, AND AN ENTIRE GENOME WOULD
ONLY COST A FEW THOUSAND DOLLARS AT THIS TIME.
• THE TECHNOLOGY IS EXPECTED TO CONTINUE TO IMPROVE IN THE FUTURE. NEW METHODS ARE
STILL UNDER DEVELOPMENT, INCLUDING SOME THAT UTILIZE NANOPORES TO SEQUENCE THE DNA.
• THIS WOULD WORK BY THREADING SINGLE STRANDS OF DNA THROUGH NANOPORES IN THE
CELL MEMBRANE, WHICH WOULD THEN BE READ BY THE TECHNOLOGY IN SINGLE FILE.
• HTTPS://NANOPORETECH.COM/APPLICATIONS/DNA-NANOPORE-SEQUENCING
 TYPES OF DNA SEQUENCING

• BROADLY SPEAKING, THERE ARE TWO TYPES OF DNA SEQUENCING: SHOTGUN AND HIGH-
THROUGHPUT.
• SHOTGUN (SANGER) SEQUENCING IS THE MORE TRADITIONAL APPROACH, WHICH IS DESIGNED FOR
SEQUENCING ENTIRE CHROMOSOMES OR LONG DNA STRANDS WITH MORE THAN 1000 BASE PAIRS. IT
INVOLVES A RAPIDLY EXPANDING FIRING PATTERN TO READ THE DNA IN SHORT FRAGMENTS OF 100 TO
1000 BASE PAIRS, WHICH ARE THEN OVERLAPPED WITH A COMPUTED ANALYSIS SYSTEM.

• HIGH-THROUGHPUT IS THE NEXT-GENERATION METHOD OF DNA SEQUENCING, WHICH HAS LED TO THE
RAPID ACCELERATION OF DNA SEQUENCING AND BROADENED KNOWLEDGE IN THE FIELD. IT IS ABLE TO
PRODUCE THOUSANDS OF SEQUENCES SIMULTANEOUSLY, WHICH LOWERS THE COST OF THE
TECHNIQUE SIGNIFICANTLY.
 WHOLE GENOME SEQUENCING AND
SEQUENCE ASSEMBLY
• A DNA SEQUENCING REACTION PRODUCES A SEQUENCE THAT IS SEVERAL HUNDRED BASES
LONG. GENE SEQUENCES ARE TYPICALLY THOUSANDS OF BASES LONG. THE LARGEST
KNOWN GENE IS THE ONE ASSOCIATED WITH DUCHENNE MUSCULAR DYSTROPHY. IT IS
APPROXIMATELY 2.4 MILLION BASES IN LENGTH. IN ORDER TO STUDY ONE WHOLE GENE,
SCIENTISTS USE A SIMPLE STRATEGY KNOWN AS SHOTGUN SEQUENCING. THE LONG DNA
SEQUENCE IS ASSEMBLED FROM A SERIES OF SHORTER OVERLAPPING SEQUENCES. LET’S SEE
WHAT HAPPENS IN THE SHOTGUN SEQUENCING APPROACH.
 SHOTGUN SEQUENCING

• SPECIAL MACHINES, KNOWN AS SEQUENCING MACHINES ARE USED TO EXTRACT SHORT

RANDOM DNA SEQUENCES FROM A PARTICULAR GENOME WE WISH TO DETERMINE (TARGET
GENOME).
• CURRENT DNA SEQUENCING TECHNOLOGIES CANNOT READ ONE WHOLE GENOME AT ONCE.
IT READS SMALL PIECES OF BETWEEN 20 AND 30000 BASES, DEPENDING ON THE
TECHNOLOGY USED. THESE SHORT PIECES ARE CALLED READS.
• SPECIAL SOFTWARE ARE USED TO ASSEMBLE THESE READS ACCORDING TO HOW
THEY OVERLAP, IN ORDER TO GENERATE CONTINUOUS STRINGS CALLED CONTIGS. THESE
CONTIGS CAN BE THE WHOLE TARGET GENOME ITSELF, OR PARTS OF THE GENOME.
• THE PROCESS OF ALIGNING AND MERGING FRAGMENTS FROM A LONGER
DNA SEQUENCE, IN ORDER TO RECONSTRUCT THE ORIGINAL SEQUENCE IS
KNOWN AS SEQUENCE ASSEMBLY.
• IN ORDER TO OBTAIN THE WHOLE GENOME SEQUENCE, WE MAY NEED TO
GENERATE MORE AND MORE RANDOM READS, UNTIL THE CONTIGS MATCH TO
THE TARGET GENOME.
SEQUENCE ASSEMBLY PROBLEM
• THIS PROBLEM IS FURTHER COMPLICATED DUE TO THE EXISTENCE OF REPETITIVE
SEQUENCES IN THE GENOME AS WELL AS SUBSTITUTIONS OR MUTATIONS
WITHIN THEM.
• THE SEQUENCE ASSEMBLY PROBLEM CAN BE COMPARED TO A REAL LIFE
SCENARIO AS FOLLOWS.
• ASSUME THAT YOU TAKE MANY COPIES OF A BOOK, PASS EACH OF THEM
THROUGH A SHREDDER WITH A DIFFERENT CUTTER, AND THEN YOU TRY TO
MAKE THE TEXT OF THE BOOK BACK TOGETHER JUST BY GLUING TOGETHER
THE SHREDDED PIECES. IT IS OBVIOUS THAT THIS TASK IS PRETTY DIFFICULT.
FURTHERMORE, THERE ARE SOME EXTRA PRACTICAL ISSUES AS WELL. THE
ORIGINAL COPY MAY HAVE MANY REPEATED PARAGRAPHS, AND SOME
SHREDS MAY BE MODIFIED DURING SHREDDING TO HAVE TYPOS. PARTS FROM
ANOTHER BOOK MAY HAVE ALSO BEEN ADDED IN, AND SOME SHREDS MAY BE
COMPLETELY UNRECOGNIZABLE.
• IT SOUNDS VERY CONFUSING AND QUITE IMPOSSIBLE TO BE CARRIED OUT. THIS PROBLEM IS
KNOWN TO BE NP COMPLETE. NP COMPLETE PROBLEMS ARE PROBLEMS WHOSE STATUS IS
UNKNOWN.
• NO POLYNOMIAL TIME ALGORITHM HAS YET BEEN DISCOVERED FOR ANY NP COMPLETE
PROBLEM, NOR HAS ANYBODY YET BEEN ABLE TO PROVE THAT NO POLYNOMIAL-TIME
ALGORITHM EXISTS FOR ANY OF THEM.
• HOWEVER, THERE ARE GREEDY ALGORITHMS TO SOLVE THE SEQUENCE ASSEMBLY PROBLEM,
WHERE EXPERIMENTS HAVE PROVEN TO PERFORM FAIRLY WELL IN PRACTICE.
• A COMMON METHOD USED TO SOLVE THE SEQUENCE ASSEMBLY PROBLEM AND PERFORM
SEQUENCE DATA ANALYSIS IS SEQUENCE ALIGNMENT.
 SEQUENCE ALIGNMENT

• SEQUENCE ALIGNMENT IS A METHOD OF ARRANGING SEQUENCES OF DNA,

RNA, OR PROTEIN TO IDENTIFY REGIONS OF SIMILARITY. THE SIMILARITY BEING
IDENTIFIED, MAY BE A RESULT OF FUNCTIONAL, STRUCTURAL, OR
EVOLUTIONARY RELATIONSHIPS BETWEEN THE SEQUENCES.
• IF WE COMPARE TWO SEQUENCES, IT IS KNOWN AS PAIRWISE SEQUENCE
ALIGNMENT. IF WE COMPARE MORE THAN TWO SEQUENCES, IT IS KNOWN
AS MULTIPLE SEQUENCE ALIGNMENT.
 NEXT GENERATION SEQUENCING

• NEXT-GENERATION SEQUENCING (NGS), ALSO KNOWN AS HIGH-THROUGHPUT SEQUENCING, IS

THE COLLECTIVE TERM USED TO DESCRIBE MANY DIFFERENT MODERN SEQUENCING
TECHNOLOGIES SUCH AS,
• ILLUMINA (SOLEXA) SEQUENCING
• ROCHE 454 SEQUENCING
• ION TORRENT PROTON / PGM SEQUENCING
• SOLID SEQUENCING
• THESE RECENT TECHNOLOGIES ALLOW US TO SEQUENCE DNA AND RNA MUCH MORE QUICKLY
AND CHEAPLY THAN THE PREVIOUSLY USED SANGER SEQUENCING, AND HAVE REVOLUTIONIZED
THE STUDY OF GENOMICS.
 APPLICATIONS OF DNA SEQUENCING

• UNDERSTANDING THE SEQUENCES OF DNA CAN BE APPLIED IN VARIOUS SETTINGS. IT NOW

FORMS THE BASE OF BIOLOGIC RESEARCH AND IS APPLIED IN BIOTECHNOLOGY, FORENSIC
BIOLOGY, VIROLOGY AND MEDICAL DIAGNOSES.
• RESEARCHERS ARE ALREADY ABLE TO USE THE RESULTS OF DNA SEQUENCING TO COMPARE
LONG LENGTHS OF DNA. IN SOME CASES, THIS MAY INCLUDE LOOKING AT SEGMENTS OF
OVER A MILLION BASES TO COMPARE DIFFERENCES IN THE SEQUENCING. THIS INFORMATION
CAN REVEAL IMPORTANT INFORMATION ABOUT THE ROLE OF CERTAIN DNA PATTERNS AND
SUSCEPTIBILITY TO HEALTH CONDITION OR RESPONSE TO MEDICAL TREATMENT.
• THE ROUTINE USE OF DNA SEQUENCING AS A DIAGNOSTIC TOOL FOR THE GENERAL
PRACTITIONER REMAINS A POSSIBILITY FOR THE FUTURE, BUT THERE ARE SOME WAYS THAT
SEQUENCING IS ALREADY BEING USED FOR MEDICAL PURPOSES. FOR EXAMPLE, DNA
SEQUENCING IS CURRENTLY USED FOR CANCER PATIENTS TO HELP IDENTIFY THE TYPE OF
CANCER THAT IS PRESENT, WHICH DIRECTS THE TREATMENT DECISIONS FOR THE PATIENT.
SIMILAR METHODS ARE CURRENTLY IN DEVELOPMENT FOR OTHER HEALTH CONDITIONS THAT
ARE LIKELY TO HAVE A GENETIC ELEMENT, SUCH AS CARDIOVASCULAR DISEASE AND DIABETES.