human genome. human genome contents: 3200 mb genes: 1200 mb –genes 48 mb –related 1152 mb:...

Human Genome

Human Genome Contents: 3200 Mb

• Genes: 1200 Mb– Genes 48 Mb– Related 1152 Mb: Pseudogenes, Gene Fragments,

Introns

• Intergenic DNA 2000 Mb– Interspersed Repeats 1400 Mb– Microsatellite (short tandem repeats) 90 Mb

• Telomeres: End Sequences• Centromeres:• Single Nucleotide Polymorphisms

Chromosomes

• Shorter than DNA they contain

• Histones: DNA binding proteins

• Two Copies held together by centromeres

• Telomere: Terminal region

• Two humans differ by 0.1%

Donors

• HGP: – Opportunity advertised near labs

– First come; First Taken

– 5-10 samples for every one used

– No link between donor and sample

• Celera: 5 subjects (three men; two women)– One Asian; One African-American; One Hispanic; Two

Caucasians

– Craig Venter

Basic Technology

• Physical Mapping

• Cloning

• Shotgun Sequencing

• Computational Sequence Reassembly

STS

• High Resolution, Rapid, Simple

• 100 - 500 bp

• Collection of overlapping fragments

• Each point represented multiple times in random fragments

• Sequence must be known

• Unique in chromosome under study

Physical Mapping

• A set of clone fragments whose position relative to each other is known

• Restriction Maps: Relative locations of Restriction Sites• Fluorescent in situ hybridization (FISH): Marker

locations mapped by hybridizing probe to chromosomes• Sequence Tagged Sites (STS): Positions of short

sequences mapped by PCR or hybridization analysis of genome fragments

• Expressed Sequence Tags (EST): short sequences from cDNA clones

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Genome cut into fragments





Cloned as library in vector (red)

Hybridisation mapping:1 pick clones into a grid 2 hybridise to probe 1 3 hybridise to probe 2 4 build contigs In this case, two clones hybridised to both probes and thus they are predicted to overlap. Those hybridising to only one probe are predicted to extend out to the left or right.




are needed to see this picture.QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.



Overlap by sharedbands

Fingerprinting:Digest clones and runOn gel

Assembly of Contiguous DNA Sequence

• Shotgun Approach

• Contigs: Result of joining overlapping sequences

• Scaffold: Result of connecting contigs by filling in gaps

• BAC: Bacteria artificial chromosome vector: Inserts 100 - 200 kbs

Regional mapping

Minimal tiling path selected for sequencing.

Regional mapping

>20 kbp

~300 bp

Molecular weightmarker every

5th laneRestriction fragmentfingerprinting

- BAC clones are grown

in 96-well format

- Hind III digest

- 1% agarose

Contig assembly

Clone A B C D E F G

FPC* Overlap identification by

restriction pattern similarities Facilitated contig assembly

*Sanger Centre C. Soderlund, I Longden and R. Mott

*

*

*

*

*

*

All restriction fragments withina clone selected for the tilingpath must be verified by theirpresence in overlapping clones. : vector fragments

: insert fragments

BCM-BCM-HGSCHGSC

Shotgun Sequencing I :RANDOM PHASE

Bac Clone: Bac Clone: 100-200 kb100-200 kb

Sheared DNA: Sheared DNA: 1.0-2.0 kb1.0-2.0 kb

SequencingSequencingTemplates: Templates:

RandomRandomReadsReads

BCM-BCM-HGSCHGSC

Shotgun Sequencing II:ASSEMBLY

ConsensusConsensusSequenceSequence

GapGap

Low Base Low Base QualityQuality

SingleSingleStrandedStrandedRegionRegion

Mis-AssemblyMis-Assembly

((InvertedInverted))

BCM-BCM-HGSCHGSC


GapGap





BCM-BCM-HGSCHGSC

Shotgun Sequencing III: FINISHING


GapGap




BCM-BCM-HGSCHGSC



GapGap



BCM-BCM-HGSCHGSC


ConsensusConsensusMis-AssemblyMis-Assembly


BCM-BCM-HGSCHGSC


BCM-BCM-HGSCHGSC


High Accuracy Sequence:High Accuracy Sequence:< 1 error/ 10,000 bases< 1 error/ 10,000 bases

Whole Genome Shotgun Sequencing

Whole Genome: Whole Genome: 3,000 Mb3,000 Mb

Sheared DNA: Sheared DNA: 1.0-2.0 kb1.0-2.0 kb

SequencingSequencingTemplates: Templates:

RandomRandomReadsReads

BCM-BCM-HGSCHGSC

Whole Genome Shotgun Sequencing:Assembly


GapGap





BCM-BCM-HGSCHGSC

Whole Genome Shotgun Sequencing:Assembly


GapGap


BCM-BCM-HGSCHGSC



Random fragmentation of genome produces good sampling of itssequence space. Overlaps are identified, and subassembly of sequence takes place after cloning into universal vector.



Digested into RandomFragments



Cloned into Vector



Sequenced from know ends of plasmid (vector)



Assembled into contigs. Gaps and single-stranded regions identified for further study. Targeted fornew sequencing. Double-Barreled: Both Strands.



In the gaps:

Whole-Genome Shotgun Sequencing

• Speed-up: Assembled Correctly?• Avoid up-front mapping• Huge amount of computer time to identify

overlaps• Have to reference a map• Repeats are a problem:

– Leave out sequence between repeats– Missing Reference End Sequence means Error

HGP

• Isolate large fragments in BACs with framework of landmark-based physical map

• Sequence on clone-by-clone basis

• Time-Consuming subcloning of random fragments and physical mapping

Sequence Reassembly

• Phrap

• Shortest Covering Superstring

• Map Assembly

• Overlap: Finding overlapping fragments

• Layout: ordering fragments

• Consensus: Sequences from layout

human genome. human genome contents: 3200 mb genes: 1200 mb –genes 48 mb –related 1152 mb:...

Documents

agarose slide

study slide

gel slide

kbs slide

regional mapping slide

human genome slide

vector red slide

randomreads bcmhgsc