NGS NEXT GENERATION SEQUENCING
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1 NGS NEXT GENERATION SEQUENCING Paestum (Sa) maggio 2014 Relatore Dr Cataldo Senatore Dr.ssa Emilia Vaccaro
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4 Sanger Sequencing Reactions For given template DNA, it s like PCR except: Uses only a single primer and polymerase to make new ssdna pieces. Includes regular nucleotides (A, C, G, T) for extension, but also includes dideoxy nucleotides. A A A A A A A G A T C C C C C C C T T T T T G G G G G G Regular Nucleotides Dideoxy Nucleotides A A A A A T C C C T T T T G G G G G 1. Labeled 2. Terminators
5 Primer T G C G C G G C C C A Sanger Sequencing 3 A C G C G C C G G G T???????????????
6 Sanger Sequencing Primer T G C G C G G C C C A G T C T T G G G C T A C G C G C C G G G T C A G A A C C C G A T C G C G 3
7 Sanger Sequencing Primer T G C G C G G C C C A G T C T T G G G C T A G C G C A C G C G C C G G G T C A G A A C C C G A T C G C G 3 T G C G C G G C C C A G T C T T G G G C T 21 bp
8 Sanger Sequencing Primer T G C G C G G C C C A G T C T T G G G C T A A C G C G C C G G G T C A G A A C C C G A T C G C G 3 T G C G C G G C C C A G T C T T G G G C T 21 bp T G C G C G G C C C A G T C T T G G G C T A G C G C 26 bp
9 Sanger Sequencing Primer T G C G C G G C C C A G A C G C G C C G G G T C A G A A C C C G A T C G C G 3 T G C G C G G C C C A G T C T T G G G C T 21 bp T G C G C G G C C C A G T C T T G G G C T A G C G C 26 bp T G C G C G G C C C A G T C T T G G G C T A 22 bp
10 Sanger Sequencing Primer T G C G C G G C C C A G T C T T G G G C A C G C G C C G G G T C A G A A C C C G A T C G C G 3 T G C G C G G C C C A G T C T T G G G C T 21 bp T G C G C G G C C C A G T C T T G G G C T A G C G C 26 bp T G C G C G G C C C A G T C T T G G G C T A 22 bp T G C G C G G C C C A G 12 bp
11 Sanger Sequencing Primer T G C G C G G C C C A G T C T T A C G C G C C G G G T C A G A A C C C G A T C G C G 3 T G C G C G G C C C A G T C T T G G G C T 21 bp T G C G C G G C C C A G T C T T G G G C T A G C G C 26 bp T G C G C G G C C C A G T C T T G G G C T A 22 bp T G C G C G G C C C A G 12 bp T G C G C G G C C C A G T C T T G G G C 20 bp
12 Sanger Sequencing 3 A C G C G C C G G G T C A G A A C C C G A T C G C G T G C G C G G C C C A G T C T T G G G C T 21 bp T G C G C G G C C C A G T C T T G G G C T A G C G C 26 bp T G C G C G G C C C A G T C T T G G G C T A 22 bp T G C G C G G C C C A G 12 bp T G C G C G G C C C A G T C T T G G G C 20 bp T G C G C G G C C C A G T C T T 16 bp
13 Sanger Sequencing 3 A C G C G C C G G G T??????????????? T G C G C G G C C C A????????? T 21 bp T G C G C G G C C C A?????????????? C 26 bp T G C G C G G C C C A?????????? A 22 bp T G C G C G G C C C A G 12 bp T G C G C G G C C C A???????? C 20 bp T G C G C G G C C C A???? T 16 bp
14 Laser Reader Sanger Sequencing T G C G C G G C C C A G T C T T G G G C T A bp
15 Sanger Sequencing Output Each sequencing reaction gives us a chromatogram, usually ~ bp:
16 Central Dogma of Molecular Biology James Watson version DNA RNA Protein So once we have the genomic DNA sequence of a species we have all of the information there is? Really?
17 No, not really.
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19 Overview The Past: Sanger The Present: Next-Gen (454, Illumina, ) The Future:? (Nanopore, MinION, Single-molecule)
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21 NGS Platforms Differ in design and chemistries Fundamentally related-sequencing of thousands to millions of clonally amplified molecules in a massively parallel manner Orders of magnitude more information-will continue to evolve Attractive for clinical applications individual sequencing assays costly and laborious- serial gene by gene analysis
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23 Shotgun sequencing by Ion Torrent Personal Genome Machine and 454
24 Shotgun sequencing by PGM/454 Genomic Fragment Adapters
25 Shotgun sequencing by PGM/454 Genomic Fragment Barcode
26 Shotgun sequencing by PGM/454
27 Multiplex Identifier Basics What is it? Two new kits, each with 6 different library adapters (total of 12 adapters) Each MID library adapter has an added, specially encoded 10-base region Used to bar-code up to 12 different genomic library samples to be run in the same region of a single sequencing run Standard Library MID Library Seq. primer Primer A Key Library fragment Primer B #bases: 40 4 Seq. primer Read Read Primer A Key MID 1 Library fragment Primer B #bases: Primer A Key MID 2 Library fragment Primer B Primer A Key MID n Library fragment Primer B CSB2008 August 2008 UCSC Sequencing Center
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32 Sequencing Cost Date Cost per Mb Cost per Genome Sep-01 $5, $95,263,072 Sep-02 $3, $61,448,422 Oct-03 $2, $40,157,554 Oct-04 $1, $18,519,312 Oct-05 $ $13,801,124 Oct-06 $ $10,474,556 Oct-07 $ $7,147,571 Oct-08 $3.81 $342,502 Oct-09 $0.78 $70,333 Oct-10 $0.32 $29,092 Oct-11 $0.09 $7,743 Oct-12 $0.07 $6,618 Jan-13 $0.06 $5,671 Source - NHGRI :
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34 Applications of Next-Generation Sequencing
35 ncrna presence in genome difficult to predict by computational methods with high certainty because the evolutionary diversity Detecting expression level changes that correlate with changes in environmental factors, with disease onset and progression, complex disease set or severity Enhance the annotation of sequenced genomes (impact of mutations more interpretable)
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38 Characterizing the biodiversity found on Earth The growing number of sequenced genomes enables us to interpret partial sequences obtained by direct sampling of specif environmental niches. Examples: ocean, acid mine site, soil, coral reefs, human microbiome which may vary according to the health status of the individual
39 Common variants have not yet completly explained complex disease genetics rare alleles also contribute Also structural variants, large and small insertions and deletions Accelerating biomedical research
40 Enable of genome-wide patterns of methylation and how this patterns change through the course of an organism s development. Enhanced potential to combine the results of different experiments, correlative analyses of genome-wide methylation, histone binding patterns and gene expression, for example.
41 Extreme example: multiplexing the amplification of human exons using primers from a programmable microarray and sequencing them using NGS.
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43 Degraded state of the sample mitdna sequencing Nuclear genomes of ancient remains: cave bear, mommoth, Neanderthal (10 6 bp ) Problems: contamination modern humans and coisolation bacterial DNA
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46 Sanger Sequencing Output Each sequencing reaction gives us a chromatogram, usually ~ bp:
47 Raw sequence data must undergo several analysis steps Preprocessing to remove adapter sequences and lowquality reads Alignment to a reference sequence or de novo alignment Analysis of compiled sequence Data Analysis
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56 L infettivologia del 3 millennio: AIDS ed altro VI Convegno Nazionale maggio 2014 Who s next 1971 Centro Congressi Hotel Ariston Paestum (SA)
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