Bassem M. Shoucri Bio Sci D145 January 8, 2015

Why do we want to map genomes? Clinical applications! – Identify genes causing diseases – Treatment? Compare genomes of different species Understand overall genome structure Understand relationship between genes and regulatory elements Map vs. sequence

Classic linkage mapping “distance” on map is actually recombination frequency (cM)

During meiosis – Recombination (meiosis I) – Segregation (meiosis II)

HAPPY Mapping: HAPloid equivalents of DNA and the PolYmerase chain reaction Mapping method that emulates recombination and segregation in vitro Why? – Eliminate need for in vivo experiments – Control over frequency of breakage – Claim more feasible than existing methods

Recombination Segregation Meiosis HAPPY

Maps need markers “a marker can be any single-copy sequence that can be amplified using the PCR to give copies that can be identified” Examples: – Sequence-tagged sites (STS) – Variable number tandem repeats (VNTR) – Single nucleotide polymorphisms (SNP) – Restriction fragment length polymorphism (RFLP)

Isolate genomic DNA Approach Fragment randomly Dilute haploid equivalents Assess linkage, assign LOD Create map

Methods Mapped 7 markers at the DMD locus (1.24 Mbp) on the X chromosome Lymphocytes from female blood  captured in agarose beads (3 ug DNA/mL packed beads) Break DNA by γ-irradiation or shearing

Methods cont’ Load DNA onto gel for Pulse-field gel electrophoresis (PFGE) Extract DNA of appropriate size – 1.75, 2.75 Mbp from γ- irradiated samples – 0.05 – 0.4 Mbp from sheared samples

Mapping panel 2-phase or nested PCR – Reduce nonspecific products First external primers (9-EXT) then internal primers (9-INT) Also carried out whole genome amplification (via random 15-mers) and 2-phase PCR

Assessing linkage LOD score: probability the markers are linked. Logarithm of the ODds > 3 (1000:1) is evidence for linkage Θ, recombination fraction: probability there is breakage between markers. Maximum Θ of 1 = complete breakage

LOD, Θ for 1.75 Mbp map

Compare LOD, Θ with known inter-marker distances! Large fragments allow for reliable mapping over greater distances Small fragments allow for greater resolution

HAPPY Maps! Brackets indicate uncertain order Sheared fragments (not shown) can map small distances (D31-D32) Known map 1.75 Mbp 2.75 Mbp WG 1.75 Mbp

Discussion, Conclusions in vitro approach to linkage mapping using a clever analog of meiosis Pros – Control frequency of breakage – Can use any marker – No cloning required Cons – Must know something about sequence – Whole genome amplification is limiting

“Up to date, only eight maps have been generated using the HAPPY approach and all of them were contributed by the inventors’ group [Dear and Cook].”

Why so SAD? Pros – Control frequency of breakage – Can use any marker – No cloning required Cons – Must know something about sequence X. tropicalis genome sequenced but NOT assembled – Whole genome amplification is limiting Material, coverage, amplification bias Resolve with Multiple displacement amplification (MDA, Φ29 polymerase)  SNPs, genes w/ human analogs

Frogs?? Study of vertabrate embryogenesis X. tropicalis – Only diploid Xenopus – 10 chromosome pairs – Sequenced, not assembled

Sequencing will only get you so far… Contig is a continuous stretch of gDNA in which the sequence is known with high confidence genome.jgi.doe.gov

Assembling the genome 90% X. tropicalis genome are contigs (1.33Gbp), 10% gaps – Genome is complex with many scattered repeats Interspersed gaps interfere with understanding of complete genome

Methods are essentially the same EXCEPT – SNPs as markers – MDA for genome wide amp. Jiang et al. Int J Biol Sci

MDA A.Primers are bound B.Φ29 DNA Pol elongates, strands are displaced C.Displaced strands can also serve as primer templates D.LOTS of material is generated Lovmar et al. Hum Mutat

It worked!

Ultra-conserved elements Regions of DNA that are conserved in at least 2 species (H. sapiens, X. tropicalis) Ni et al. Genes Dev

HAPPY Mapping of X. Tropicalis using SNPs within ultra-conserved elements

Further reading… Jiang, Rokhsar, Harland. Int J Biol Sci. 2009; 5(6): 621. Acknowledgment – Dr. Amanda Janesick