PowerPoint Presentation
NEXT GENERATION SEQUENCINGPresented byDr. Rayhan ShahrearMS
ResidentPhase A, Year 2, Block 6Dept. of Anatomy, BSMMU.Guided
byProf. Laila Anjuman BanuChairman and ProfessorMolecular Biology
and GeneticsDept. of Anatomy, BSMMU.
ObjectivesAudience will be able to-define DNA sequencingdescribe
the history of DNA sequencingdescribe the steps of Sanger
sequencingdefine next generation sequencingdescribe the steps of
next generation DNA sequencingdescribe use of DNA sequencing
What is DNA sequencing?Sequencing DNA means determining the
order of the four chemical building blocks - called "bases" - that
make up the DNA molecule.
History of DNA Sequencing1953 - Watson and Crick1965 - Robert
Holley1970 - Ray WU1977 - Fredric Sanger and Maxam-Gilbert1986 -
Semi-automated DNA sequencing machine1990 - Step by step
sequencing1996 - Pyrosequencing1997 - DNA colony sequencing1998 -
Phred quality score2000 - Massively parallel signature sequencing
2004 - Massively parallel pyrosequencing
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SANGER SEQUENCING
1977
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BILLIONS
dNTPs +Primers +EnzymesddNTPsddATPsddTTPsddCTPsddGTPs
TAGATGCTGddATPsddTTPsddCTPsddGTPs
NEXT GENERATION SEQUENCING
What is NGS?Next-generation sequencing (NGS), also known as
high-throughput sequencing is the catch-all term used to describe a
number of different modern sequencing
technologies.E.g.PyrosequencingIllumina sequencingSOLiD
sequencingIon Torrent semiconductor sequencing etc.
Illumina sequencing
StepsCreating DNA librariesTagmentationIndexingCleaning
upNormalizationSequence by synthesisLoading flow
cellAmplificationSequencingAnalysis of the sequenced data
But
DNA Extraction
Quality and Quantity Check
PCR
Amplicon Quantity Check
Creating DNA library
CleavingTaggingIndexing
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A1B1C1D1E1F1G1H1A2B2C2D2E2F2G2H2A3B3C3D3E3F3G3H3A4B4C4D4E4F4G4H4A5B5C5D5E5F5G5H5A6B6C6D6E6F6G6H6A7B7C7D7E7F7G7H7A8B8C8D8E8F8G8H8A9B9C9D9E9F9G9H9A10B10C10D10E10F10G10H10A11B11C11D11E11F11G11H11A12B12C12D12E12F12G12H12910111287654321ABCDEFGH
Sequence by SynthesisLoading flow cell
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Sequence by Synthesis
Amplification
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Sequence by SynthesisAmplification
Sequence by Synthesis
Sequencing
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Data Analysis
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Use of DNA SequencingIt helps to understand and comprehend the
internal structure of genes in the DNA.It helps to understand which
sequence codes for what kind of proteins.The knowledge of sequence
can tell if there is any disease or not.The knowledge of sequence
can be used to prepare proteins.The knowledge of sequencing will
help to cure many diseases.
Why NGS?Sequencing mechanismRead lengthAccuracyReadsOutput
data/runTime/runAdvantageDisadvantageInstrument priceLibrary
preparationCost
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NGSSanger sequencingSequencing mechanismSequencing by
synthesisDideoxy chain terminationRead length50SE, 50PE, 101PE400
900 bpAccuracy98%, (100PE)99.999%Reads30kWholeOutput
data/run600Gb1.9~84KbTime/run3~10 Days20Mins~3HoursAdvantageHigh
throughputHigh quality, long read lengthDisadvantageShort read
assemblyHigh cost low throughputInstrument priceInstrument
$690,000, $6000/(30x) human genomeInstrument $95,000, about $4 per
800bp reactionLibrary preparationYesNoCost/million
bases$0.07$2400
Thank You
