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Nextgenerationsequencing

AllenVanDeynze

UC DavisNovember16th,2010

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Marker development considerations

How to sequence?

What part of the DNA to sequence?

Talk 2

What lines to sequence?

How many lines to sequence?

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Sequencing DNA

The goal of sequencing DNA is to tellthe order of the bases, or 

nucleotides, that form the inside

of the double-helix molecule.

g roug pu sequenc ng me o s

• Sanger/Dideoxy

• 2nd generation (NextGen)

• 3rd generation

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Sanger Dideoxy DNA sequencing

650-1000 bp

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454-Pyrosequencing

Perform emulsion

PCRConstruct

Single stranded

adaptor ligatedDNA Depositing DNA Beads into the

PicoTiter™Plate

 Simultaneous sequencing of the entire genome in

hundreds of thousands of picoliter-size wells

yrop osp ate s gna generat on

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Solexa/lIlumina Sequencing

• Sequencing by synthesis (not chain termination)

 

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Helicos-True Single Molecule Sequencing (tSMS)™

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Single Molecule Real Time sequencing

10 bp/sec

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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.

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Sequencing technology 2010

MB/run Cost/MB

 

reads (bp)

Sanger  0.29 4,333 700

  . -Illumina 20,000 0.50 30-125

Illumina 2010 100,000 0.10 85-100

Helicos 500,000 0.02? 25

Ion Torrent ? ? 25

Pacific Bio 1 000 ? >1000

2010- 10-50 faster..and cheaper 

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Nextgenerationsequencing

AllenVanDeynze

UC DavisNovember16th,2010

8/6/2019 01 VanDeynze Sequencing_0

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Marker development considerations

How to sequence?

What part of the DNA to sequence?

Talk 2

What lines to sequence?

How many lines to sequence?

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Transcriptome sequencing Illumina

Library creation/QC GAII sequencing(single and paired end)Root

ea

Flower 

Fruit

Assembly

Data Collection

Analysis: transcriptome complexity

SNP calling/validation

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Sequencing all of the EST

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Sequencing beyond ESTs

Whole Genome Shotgun Sequencing

Start with a whole enome 

Shear the DNA into many different, random

se ments.

Sequence each of the random segments.

Then ut the ieces back to ether a ain in their original order using a computer 

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Anatomy of a WGS Assembly

STSSTS

Genetic and physical map

STSSTS--mapped Scaffoldsmapped Scaffolds

ContigContigPac BioSanger 

ap mean s . ev. nownap mean s . ev. nownRead pair (mates)Read pair (mates)

ConsensusConsensus

454

Illumina

Reads (of several haplotypes)Reads (of several haplotypes)

SNPsSNPs

 Helicos

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So what?

Anchored Genome Assembly

Gene function

 Gene model

Allele

Functional mutation

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Genome Browser