1. Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

2. Why This Study Is Impressive?
Lower sequencing cost + more sophisticated algorithms = more species with genome assemblies
The problem ?  These assemblies are fragmented, contain gaps and errors which make downstream applications difficult
This study  Uses combined technologies (3) that give the most continuous de novo mammalian assembly
1- Long reads for contig formation
2 – Short reads for consensus validation
3 – Scaffolding by optical and chromatin interaction mapping
400 fold improvement in continuity
Only 649 gaps
De novo = starting from the beginning
Contig = overlapping sequence data
Consensus validation = confirmation or corroboration; the declaration of validity.
Scaffolds = are composed of contigs and gaps.

3. How is This Applicable in The Real World?
Agriculture  accurate genome reference is essential for plants and animal species
Researchers of such organism
GMO = is the result of a laboratory process where genes from the DNA of one species are extracted and artificially forced into the genes of an unrelated plant or animal. The foreign genes may come from bacteria, viruses, insects, animals or even humans.

4. Current Progress of Gene Sequencing
Progress has been made in techniques to generate contig regions
Finish the genome is the challenge  extremely difficult for repetitive genomes
The human genome  Draft in 2001  followed by 3 years curation by 18 institutions
Short-read sequencing  inexpensive, yield draft genome assemblies but they are highly fragmented  (hence this paper combining 3 techniques)

5. Current Progress of Gene Sequencing
Repetition in the genome is the biggest challenge in its assembly  leads to gaps
Scaffolding technologies  Order and orient the assembly of contigs (used in this paper)
Chromosome interaction mapping  identifies long-range chromosome interactions
Optical mapping  inexpensive, HD scaffolding data
Both methods have limited ability to
scaffold small contigs in fragmented
short read assemblies
Scaffolds = are composed of contigs and gaps.

6. Current Progress of Gene Sequencing
Single-molecule sequencing  produces reads of 10’s kb but has high error rate
The Pacific Biosciences sequencing platform  produces reads at an average of 14 kb (peak over 60 kb)  used to construct bacterial and continuous eukaryotic genomes
Combination of long –read sequencing + long-range scaffolding = most efficient way to produce near-complete genome reference assemblies

7. - Combination of long –read sequencing + long-range scaffolding = most efficient way to produce near-complete genome reference assemblies

8. Online Methods Listed
Animals  Under IACUC-approved protocol and other federal regulations
Reference individual selection  DNA panel composed of 96 US goats assembled to find most homozygous goat  Determined by raw count of homozygous methods
Genome sequencing, analysis and sequencing
Conflict resolutions  To resolve misassembles in prior steps
Assembly polishing and contaminant identification
Assembly annotation
Gap resolution and repeat analysis
Centromeric and telomeric repeat analysis
Fosmid end sequencing and analysis
Statistical analysis
Code availability
Data availability
- Detailed and above undergraduate understanding, but students may refer to the online methods if they recognize these methods / steps listed and would like to know more

9. RR
Genome sequencing, analysis and sequencing
Conflict resolutions  To resolve misassembles in prior steps
Gap resolution and repeat analysis

10. Results Adult male goat (San Clemente breed) sequenced
Goat had high degree of homozygosity to minimize heterozygous alleles to simplify the genome assembly
Long-read single-molecule sequencing
High fidelity short-read sequencing
Optical mapping (scaffolding tech)
Chromatin interaction mapping (scaffolding map)
Stepwise assembly of this complementary data as observed in table 1

11. Stepwise assembly of complementary data
Validated with statistical methods

12. Research Limitations
RH mapping used to maximize the accuracy of the final reference assembly
Corrected 21 inversions  consisting of 83 scaffolds
Corrected 4 misplacements before final gap filling
ARS1  Final assembly
After error correction and validation, ARS 1 contains 4 discrepancies with the RH map  needs further research to fix these (figure 3)
ARS1 compares favorably with the human genome !
RH map = radiation hybrid mapping  technique used to map mammalian chromosomes
Uses x-ray breakage of chromosomes to determine the distances between DNA markers as well as their order on the chromosomes
ARS 1 = autonomously replicating sequence 1
ARS1
Human Genome
Scaffolds 31 24
Gaps
649
832

14. -R

15. Implications
Paper presents near-finished reference genome for the domestic goat using:
Long-read single-molecule sequencing
High fidelity short-read sequencing
Optical mapping (scaffolding tech)
Chromatin interaction mapping (scaffolding map)
Unlike cattle that come from two different subspecies, dometic goats appear to come from one single ancestor  bezoar33
This new assembly strategy is superior in accuracy and cost effectiveness compared to the past
Provides new standard reference for ruminant genetics
Creation of the reference goat genome could mean easier identification of adaptive variants in the sequence data of descendent breeds

16. Adult male San Clemente breed
Unlike cattle that come from two different subspecies, dometic goats appear to come from one single ancestor  bezoar33
This new assembly strategy is superior in accuracy and cost effectiveness compared to the past

17. Discussion Long-read sequencing  improved mammalian genome assemblies
Complex genomic regions continue to interfere with the complete assembly
Current long-read technologies  still falling short
Cannot regularly produce completely assembled chromosomes
Scaffolding technologies  Must be reliable and affordable  becomes important to generate HD, finished reference genome because current long-read is not enough
This is why this paper combined all three methods !
Demonstrated that optical + chromatin interaction mapping are complementary and useful in conjunction with long-read assemblies

18. Discussion Methods of this study reduced the cost of genome finishing
It would cause around $100,000 to perform a similar genome assembly using current PacBio RS II and the scaffolding techniques used in this study
3X cost of a short-read assembly but would provide unparallel gain in continuity and quality of the genome assembly
From this study, it is expected that these methods will allow the de novo assembly of many vertebrate species without compromising the quality !
- The Pacific Biosciences sequencing platform  produces reads at an average of 14 kb (peak over 60 kb)  used to construct bacterial and continuous eukaryotic genomes

19. Questions?

20. References
Bickhart, Derek M. et al. Single-Molecule Sequencing and Chromatin Conformation Capture Enable De Novo Reference Assembly of the Domestic Goat Genome. Nature Genetics , 6 Mar. 2017,