1. Riann Egusquiza D145 Presentation January 12, 2017
This first article I am going to present is about a method, called happy mapping, that was developed to aid in genome mapping
Riann Egusquiza
D145 Presentation
January 12, 2017

2. What is genome mapping?
The use of various methods to determine the locus of a genetic landmarks (genes, regulatory sites, etc.) and the relative distances between genetic landmarks in the genome
So as a refresher, genome mapping is…for example, a genetic map will specify where all the genes in a given chromosome are located (the locus and distance between genes are not always exact in a genetic map)

3. Why would we want to map genomes?
Compare genomes of different species
Find new genes
Identify disease causing genes
Understand overall genome structure
Understand relationship between genes and regulatory elements
Aid in assembly after sequencing
So why is mapping genomes important? Basically, the more you know the more helpful it can be for the advancement of science and medicine

4. Classical Linkage Mapping
Determines distance between genes through the rate of meiotic recombination
Drawbacks:
In vivo studies required
Natural meiotic recombination is not random
The infrequency of recombination limits the resolution
Classical linkage mapping is the original method to utilize the rate of meiotic recombination to predict the distance between genes…which also supports the idea of linked genes. Although this method is very useful, it does have some drawbacks…

5. HAPPY mapping: HAPloid equivalents of DNA and PolYmerase chain reaction
Analogous to classical linkage mapping
utilizes in vitro equivalents to meiotic recombination and division
overcomes the drawbacks of classical linkage mapping and results in a higher resolution map
The authors of this paper developed the HAPPY mapping methods to overcome these drawbacks. HAPPY stands for haploid equivalents of DNA and Polymerase chain reaction. It is….followed by the use of PCR to detect the genetic markers in each haploid equivalent

6. HAPPY mapping protocol
PCR for 7 markers
Step 1
Step 2
Step 3
γ-irradiation or shearing
In vitro method analogous to meiotic recombination
In vitro method analogous to meiotic division
Step 4

7. Meiosis HAPPY Recombination Segregation
So here I am showing the HAPPY methods side by side a diagram of meiosis so you can see how it is an in vitro equivalent. So in in meiosis, homologous chromosomes undergo recombination leading to the DNA becoming fragmented. In HAPPY mapping they perform in vitro random fragmentation of the DNA. Homologous chromosomes then undergo two division for that there is 1 haploid per cell. In happy mapping, they dilute the fragmented DNA until it is ~1 haploid per tube. PCR is then conducted on the markers of interest for all 1 tubes to determine which markers tend to be fragmented together.
HAPPY
Segregation

8. DMD locus map was already known so it was used as a proof of principle for their method, to show that happy mapping could give reliable mapping
PCR for 7 markers (D0, D3, D21, D31, D32, D45, D48) of the DMD locus of the human X chromosome
**the map of the DMD locus was already known

9. PCR of ~1 haploid aliquots using primer sets for 9 markers
Run the PCR products on a gel to determine what marks are present in each fragment
Whole genome PCR then PCR of ~1 haploid aliquots using primer sets for 9 markers
PCR of ~1 haploid aliquots using primer sets for 9 markers
Aliquot #
Whole genome DNA amplification can be used to test more markers from the same ~1 haploid sample
The PCR product of each aliquot was then run on gel to determine what markers are present in each one. As you can see some markers tend to be detected together in the same aliquots (such as D31 and D32) suggesting that they are close together
Markers that tend to show up together are most likely close in distance

10. Calculating the LOD of the markers
LOD= logarithm of the odds of linkage They are quantifying the likelihood of two markers being linked
The bigger the LOD the greater chance they are linked
Hence, closer together in the genome
LOD >3 is considered evidence of linkage

11. Using the LOD scores they generated possible marker orders
Using the LOD scores they generated possible marker orders. This method was done using DNA fragmented to ~1.75 Mbp and ~2.75 Mbp. Another assay with ~1.75 Mbp fragments were don’t, but with adding a whole-genome PCR step before the marker PCR step

12. HAPPY mapping gives accurate genome map
Known map
~1.75 Mbp
~2.75 Mbp
~1.75 Mbp w/ whole genome PCR
Comparing the HAPPY maps to the known map of the DMD locus shows high resemblance
Notes:
Overall the total map length is slightly shorter
It overestimates shorter distances

13. Additional analysis of the method
Longer fragments demonstrate higher accuracy
Shearing can be used to obtain higher resolution between small distances
Over sonication destroys linkages
Amplifying whole genome before performing PCR of makers gives similar results
Theta is another measurement that is the fraction of possible linkage. The highest value of theta being 1 representing no linkage.

14. Summary/Conclusions
Dear an Cook developed a new method, analogous to classical linkage mapping, that utilizes in vitro equivalents to meiotic recombination and division
It doesn’t require in vivo studies and bypasses the issue of non- random DNA fragmentation
HAPPY mapping is able to generate reliable genetic maps
This method gives higher resolution than classical linkage mapping
The method can be fine-tuned to produce different fragment sizes to investigate different levels of resolution
Cons:
it requires that you know something about the sequence (because you have to design the PCR primers)
Whole genome amplification is limiting

15. This next article I am going to present uses the foundation of HAPPY mapping to use for mapping the genome of Xenopus tropicalis

16. HAPPY mapping
“Up to date, only 8 maps have been generated using the HAPPY approach and all of them were contributed by the inventor’s group”
So why hasn’t HAPPY mapping been more utilized to map genomes?
Lacks the necessary amplification of whole genome DNA to provide enough starting material to detecting a large number of markers
Up to the publication of this paper (22 years after its development) only….bottleneck issue (other methods did not have this same issue)

17. New technology to revamp the HAPPY mapping technique
Multiple Displacement Amplification (MDA) can overcome the issue of lack of starting DNA material
It can yield µg of amplified DNA starting with as few as 1-10 copies of DNA
For this paper, they introduced the use of Multiple Displacement Amplification into their HAPPY mapping pipeline

18. Using HAPPY mapping to map the Xenopus tropicalis genome
Why frogs?
The study of amphibian embryogenesis provides important insight into the mechanisms underlying vertebrate development
The genome has been sequenced, but there are many gaps which has halted the assembly
The authors suggest using HAPPY approach for construction of whole genome maps to aid in a high quality long-range assembly of the X. tropicalis genome
They wanted to use HAPPY mapping to map the genome of Xenopus tropicalis because…

19. Mapping the Xenopus tropicalis genome using the HAPPY approach
HAPPY mapping method EXCEPT
MDA to amplify whole genome DNA
Markers are Ultra Conserved Elements (UCE’s) in relation to the human genome
They used basically the same protocol as the previous paper except.

20. Analyzing the new HAPPY protocol
MDA amplifies DNA yield
Retention rate= % of genes retained in the fragments
Fragment size impacts retention rate

21. Analyzing the new HAPPY protocol
Whole genome samples
(positive controls)
HAPPY lines
Average retention rate = 21.7%

22. Frog genome assembly Human chromosome 1 Their HAPPY map
Alignment of the frog happy map found 8 tentative conserved segments. Assembling using based on the comparison of the two genomes found 10. However, the marker order for the happy map is more consistent with the locations on each scaffold assembled in the previous version of the xenopus genome assembly…they believe that the happy map had superior marker order

23. Summary/Conclusions Using MDA improved the conventional HAPPY method
They developed the first HAPPY panel of Xenopus tropicalis genome using UCE’s
Established a pilot HAPPY map for Xenopus tropicalis
They suggest to replace the PCR steps with next generation sequencing to further improve the pipeline for whole genome sequencing, mapping, and assembly

24. Additional Reading

25. Important notes for your presentation
Introduction/Background: provide the necessary information to introduce the topic and help explain the purpose of the paper
Figures- know what they are saying and how the data was generated. Also, discuss how they are contributing to the hypothesis
Conclusions- list the main findings. Don’t be afraid to be critical!
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