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    01 VanDeynze Sequencing - 0

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    Francisco Fuentes
    The goal of sequencing DNA is to tell the order of the bases, or nucleotides, that form the inside of the double-helix molecule. High throughput sequencing methods Hi h th h t I th d Sanger / Dideoxy 2nd generation (NextGen) 3rd generation Sanger Dideoxy DNA sequencing Construct Single stranded adaptor ligated DNA Perform emulsion PCR Depositing DNA Beads into the PicoTit

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    01 VanDeynze Sequencing - 0

    Uploaded by

    Francisco Fuentes
    619 views23 pages
    The goal of sequencing DNA is to tell the order of the bases, or nucleotides, that form the inside of the double-helix molecule. High throughput sequencing methods Hi h th h t I th d Sanger / Dideoxy 2nd generation (NextGen) 3rd generation Sanger Dideoxy DNA sequencing Construct Single stranded adaptor ligated DNA Perform emulsion PCR Depositing DNA Beads into the PicoTit

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    01 VanDeynze Sequencing_0

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    The goal of sequencing DNA is to tell the order of the bases, or nucleotides, that form the inside of the double-helix molecule. High throughput sequencing methods Hi h th h t I th d Sanger / Dideoxy 2nd generation (NextGen) 3rd generation Sanger Dideoxy DNA sequencing Construct Single stranded adaptor ligated DNA Perform emulsion PCR Depositing DNA Beads into the PicoTit

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    619 views23 pages

    01 VanDeynze Sequencing - 0

    Uploaded by

    Francisco Fuentes
    The goal of sequencing DNA is to tell the order of the bases, or nucleotides, that form the inside of the double-helix molecule. High throughput sequencing methods Hi h th h t I th d Sanger / Dideoxy 2nd generation (NextGen) 3rd generation Sanger Dideoxy DNA sequencing Construct Single stranded adaptor ligated DNA Perform emulsion PCR Depositing DNA Beads into the PicoTit

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    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 23

    Next generation sequencing

    Allen Van Deynze UC Davis November 16th, 2010

    Marker development considerations How to sequence? q What part of the DNA to sequence? Talk 2 What lines to sequence? How many lines to sequence? y

    Sequencing DNA
    The goal of sequencing DNA is to tell the order of the bases, or nucleotides, that form the inside of the double-helix molecule. High throughput sequencing methods Hi h th h t i th d Sanger/Dideoxy 2nd generation (NextGen) 3rd generation

    Sanger Dideoxy DNA sequencing

    650-1000 bp

    454-Pyrosequencing

    Construct Single stranded adaptor ligated DNA

    Perform emulsion PCR


    Depositing DNA Beads into the PicoTiterPlate

    Sequencing by Synthesis: Simultaneous sequencing of the entire genome in hundreds of thousands of picoliter-size wells Pyrophosphate signal generation P h h t i l ti

    Solexa/lIlumina Sequencing
    Sequencing by synthesis (not chain termination) Generate up to 100 Gb per run

    Helicos-True Single Molecule Sequencing (tSMS)

    Single Molecule Real Time sequencing 10 bp/sec

    Ion Torrent

    In nature, when a nucleotide is incorporated into a strand of DNA by a polymerase, a hydrogen ion is released as a byproduct.

    Sequencing technology 2010


    Length of reads (bp) 700 175-450 175 450 30-125 85-100 25 25 >1000

    Sanger Roche 454 Illumina Illumina 2010 Helicos Ion Torrent Pacific Bio

    MB/run 0.29 180 20,000 100,000 500,000 ? , 1,000

    Cost/MB 4,333 55.56 55 56 0.50 0.10 0.02? ? ?

    2010- 10-50 faster..and cheaper

    Next generation sequencing


    Allen Van Deynze UC Davis November 16th, 2010

    Marker development considerations How to sequence? q What part of the DNA to sequence? Talk 2 What lines to sequence? How many lines to sequence? y

    Where to sequence? Eukaryotic Genomes and Gene Structures

    Gene G

    Intergenic Gene Region

    Intergenic Gene Region

    Locus/Gene Gene models Full length cDNAs Expressed Sequence Tags

    Transcriptome sequencing Illumina


    Library creation/QC Root Leaf L f Flower Fruit 350

    GAII sequencing (single and paired end)

    Assembly
    Data Collection

    Analysis: transcriptome complexity SNP calling/validation

    Sequencing all of the EST

    Sequencing beyond ESTs

    Whole Genome Shotgun Sequencing Start with a whole genome Shear the DNA into many different, random segments. g Sequence each of the random segments. Then, put the pieces back together again in their original order using a computer

    Anatomy of a WGS Assembly


    Genetic and physical map
    STS Chromosome

    STSSTS-mapped Scaffolds

    Pac Bio Sanger 454 Illumina Ion Torrent


    Helicos

    Contig Read pair (mates) Gap ( G (mean & std. d td dev. K Known) )

    Consensus Reads (of several haplotypes) SNPs

    So what?
    Anchored Genome Assembly y Gene function Gene order Gene model Allele Functional mutation

    Genome Browser

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