Whole genome sequencing and assembly of an avian genome, the European crow Corvus corone spec.

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(1)MSc BIOINF 11 001. Whole genome sequencing and assembly of an avian genome, the European crow Corvus corone spec. A reduced representation library based approach. Nagarjun Vijay Degree project in bioinformatics, 2011 Examensarbete i bioinformatik 45 hp till masterexamen, 2011 Biology Education Centre and Dept. of Evolutionary biology , EBC, Uppsala University Supervisor: Dr Jochen Wolf.

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(117) Whole genome sequencing and assembly of an avian genome, the European crow Corvus corone spec. A reduced representation library based approach -. Popular science summary. Nagarjun Vijay The complete hereditary information of an organism which mostly decides the various characters of an organism are stored in its genome in the form of different combinations of nucleotide bases in the DNA. To understand the various characters of an organism, genes and functional elements responsible for these features it is useful to sequence its genome. Sequencing involves finding the order in which the nucleotide bases are organized. Various sequencing methods can determine the order of the bases in a stretch of 100 to a maximum of 1000 bases. Hence, for organisms with large genomes it is not possible to sequence the entire genome directly.. The maximum number of consecutive bases that can be sequenced is limited to a maximum of 1.5 KB. To overcome this limitation 'shotgun' genome sequencing has been utilised. A sidewalk will eventually be completely covered by randomly falling raindrops. Similarly, the entire genome can be covered by randomly sequencing smaller fragments of the genome. This is done by first breaking down DNA into a number of random fragments of length suitable for sequencing. These fragments are then sequenced individually. Enough fragments are sequenced to have covered the genome multiple times. Sequenced pieces of the genome are put together into a single continuous sequence of DNA using a computer program called “Genome assembler”. The genome assembler looks for overlapping regions between the sequenced fragments and makes use of this information to place the different fragments with respect to each other. This method of genome assembly is known as ‘shotgun’ genome sequencing. In this project we tested the benefits of an alternative method for genome assembly called reduced representation library approach. In this approach the genome is first partitioned into smaller reproducible fractions which are then individually subjected to shotgun genome sequencing. These individually assembled genomes are then put together to obtain a complete genome assembly. Partitioning of the genome into smaller fractions is expected to reduce the number of incorrect assemblies and allow for faster assembling of the data. Thereby using in-silico simulation on the zebra finch genome the reduced representation library approach has been found to provide assemblies which are more contiguous than those obtained from shotgun genome sequencing. Degree project in bioinformatics, 2011 Examensarbete i bioinformatik 45 hp till masterexamen, 2011 Biology Education Centre and Dept. of Evolutionary biology, EBC, Uppsala University Supervisor: Dr Jochen Wolf.

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(119) ͞,ǇƉĞƌďŽůŽŝĚƐŽĨǁŽŶĚƌŽƵƐ>ŝŐŚƚ ZŽůůŝŶŐĨŽƌĂǇĞƚŚƌŽƵŐŚ^ƉĂĐĞĂŶĚdŝŵĞ ,ĂƌďŽƵƌƚŚŽƐĞtĂǀĞƐǁŚŝĐŚƐŽŵĞŚŽǁDŝŐŚƚ WůĂǇŽƵƚ'ŽĚΖƐŚŽůǇƉĂŶƚŽŵŝŵĞ͟ . . . ͲdƵƌŶŝŶŐ. . . 7.

(120) Contents 1 Abstract ...................................................................................................................................................3 2 Introduction .............................................................................................................................................9 2.1 Whole genome shotgun assembly ............................................................................................10 2.1.1 Traditional sanger sequencing ......................................................................................12 2.1.2 NGS: Next-generation sequencing ...............................................................................13 2.1.3 Genome assembly and its challenges ...........................................................................14 2.2 Partitioning the genome to reduce the problem of assembly....................................................17 2.2.1 BAC clones ............................................................................................................................17 2.2.2 Reduced representation library based genome assembly .............................................18 2.3 Sequencing the crow genome ...................................................................................................20 3 Materials and methods ..........................................................................................................................22 3.1 In silico genome sequencing.....................................................................................................22 3.1.1 Restriction enzyme and fragment size selection ..........................................................23 3.1.2 de novo assembly .........................................................................................................30 3.2 Laboratory methods ..................................................................................................................31 3.2.1 DNA extraction and quality check ...............................................................................32 3.2.2 Reduced representation library construction ................................................................33 4 Results ...................................................................................................................................................35 4.1 Simulated assemblies ................................................................................................................35 4.1.1 Comparison of WGS vs RRL strategy .........................................................................35 4.1.2 Evaluation of RRL library preparation in the laboratory .............................................35 5 Discussion .............................................................................................................................................38 6 Conclusion ............................................................................................................................................41 7 References .............................................................................................................................................43 8 Supplementary material ........................................................................................................................49 8.1 Source code for various scripts used in the project ..................................................................49 8.2 DNA extraction from blood (minimal fragmentation) .............................................................69 8.3 Reduced representation library construction ............................................................................71 8.4 List of figures............................................................................................................................75 8.5 List of tables .............................................................................................................................76 8.6 List of Acronyms ......................................................................................................................77 9 Acknowledgments ...............................................................................................................................78. 8.

(121) 2 Introduction ǇƚŚĞƚƵƌŶŽĨƚŚĞĐĞŶƚƵƌǇ͕EƐĞƋƵĞŶĐŝŶŐŚĂĚƐĐĂůĞĚƵƉƌĞŵĂƌŬĂďůǇĂŶĚƚŚĞƉƵďůŝƐŚŝŶŐŽĨ ƚŚĞŚƵŵĂŶŐĞŶŽŵĞŝŶϮϬϬϭŚĞƌĂůĚĞĚƚŚĞƉŽƐƚͲŐĞŶŽŵŝĐĞƌĂ;sĞŶƚĞƌĞƚĂů͕͘ϮϬϬϭͿ͘ĞŝŶŐĂďůĞ ƚŽƌĞĂĚŐĞŶŽŵĞƐŚĂƐŚĞůƉĞĚƵƐƵŶĚĞƌƐƚĂŶĚƚŚĞĨƵŶĐƚŝŽŶĂůĂƐƉĞĐƚƐŽĨŐĞŶĞƐĂŶĚƌĞŐƵůĂƚŽƌǇ ŶĞƚǁŽƌŬƐ;ŶŶĂ^ŚĂƌŵĂŶĞƚĂů͕͘ϮϬϬϭͿ͘^ĞƋƵĞŶĐŝŶŐŽĨŵƵůƚŝƉůĞĐůŽƐĞůǇƌĞůĂƚĞĚŽƌŐĂŶŝƐŵƐŚĂƐ ĂůůŽǁĞĚ ĨŽƌ ƚŚĞ ĐŽŵƉĂƌĂƚŝǀĞ ĂŶĂůǇƐĞƐ ŽĨ ŐĞŶŽŵĞƐ ǁŚŝĐŚ ŝƐ ǀĞƌǇ ƵƐĞĨƵů ŝŶ ĞǀŽůƵƚŝŽŶĂƌǇ ƐƚƵĚŝĞƐ͘ tŚŝůĞ ŵŽƐƚ ŽĨ ƚŚĞ ŵĂŶƵĂůůǇ ŵĂŝŶƚĂŝŶĞĚ ŐĞŶŽŵĞƐ ĂǀĂŝůĂďůĞ ŶŽǁ ŚĂǀĞ ďĞĞŶ ƉƌŽĚƵĐĞĚ ǁŝƚŚ ƚŚĞ ĚŝͲĚĞŽǆǇ ĐŚĂŝŶ ƚĞƌŵŝŶĂƚŝŽŶ ŵĞƚŚŽĚ͕ ƐĞǀĞƌĂů ŶŽǀĞů ŵĞƚŚŽĚƐ ŽĨ E ƐĞƋƵĞŶĐŝŶŐ ŚĂǀĞ ďĞĞŶ ĚĞǀĞůŽƉĞĚ ŝŶ ƐƵďƐĞƋƵĞŶƚ ĚĞĐĂĚĞƐ ;DŝĐŚĂĞů >͘ D Ğƚ Ăů͕͘ ϮϬϭϬͿ͘ /ŵƉƌŽǀĞŵĞŶƚƐŝŶƐĞƋƵĞŶĐŝŶŐƚĞĐŚŶŽůŽŐǇŚĂǀĞŵĂĚĞŝƚƉŽƐƐŝďůĞƚŽƐĞƋƵĞŶĐĞůĂƌŐĞƌŐĞŶŽŵĞƐ ĂƚůŽǁĞƌĐŽƐƚƐ͘dŚŝƐŶŽǁŽƉĞŶƐƵƉƚŚĞƉŽƐƐŝďŝůŝƚǇƚŽŝŶǀĞƐƚŝŐĂƚĞŽƌŐĂŶŝƐŵƐǁŚĞƌĞŽŶůǇƐĐĂƌĐĞ ŐĞŶĞƚŝĐĚĂƚĂŝƐĂǀĂŝůĂďůĞ͘ tŚŝůĞƚĞĐŚŶŝĐĂůůǇŶŽǁŝƚŝƐƉŽƐƐŝďůĞƚŽƉƌŽĚƵĐĞŵƵůƚŝƉůĞĨŽůĚĐŽǀĞƌĂŐĞŽĨĂŐĞŶŽŵĞƵƐŝŶŐƚŚĞ ůĂƚĞƐƚ ƐĞƋƵĞŶĐŝŶŐ ƚĞĐŚŶŽůŽŐǇ͕ Ɛƚŝůů ŵĂŶǇ ĂůŐŽƌŝƚŚŵŝĐ ĂŶĚ ĐŽŵƉƵƚĂƚŝŽŶĂů ĐŚĂůůĞŶŐĞƐ ŝŶ ŽďƚĂŝŶŝŶŐĂĨƵůůǇĂƐƐĞŵďůĞĚŚŝŐŚƋƵĂůŝƚǇĚƌĂĨƚƌĞŵĂŝŶ;^ĂŶƚĞĞƚĂů͕͘ϮϬϬϭ͕DŝůůĞƌĞƚĂů͕͘ϮϬϭϬͿ͘ tĞǁŝůůŚĞƌĞĨŝƌƐƚƌĞǀŝĞǁƚŚĞŵŽƐƚĐŽŵŵŽŶt'^ĂƉƉƌŽĂĐŚĂŶĚƚŚĞŶĚĞƐĐƌŝďĞĂǀĂƌŝĂŶƚ;ZZ>Ϳ ƚŚĂƚ ǁŝůů ďĞ ĞǆƉůŽƌĞĚ ŝŶ ŵŽƌĞ ĚĞƚĂŝů͘ dŚĞ Ăŝŵ ŽĨ ƚŚŝƐ ƐƚƵĚǇ ǁĂƐ ƚŽ ĐŽŵƉĂƌĞ ƚŚĞ ƚǁŽ ǁŝƚŚ ƌĞƐƉĞĐƚ ƚŽ ĐŽŶƚŝŐƵŝƚǇ͕ ĂĐĐƵƌĂĐǇ͕ ƌĞĐŽǀĞƌǇ ĂŶĚ ŝŶĐůƵĚĞ ƐŽŵĞ ĞǆƉĞƌŝŵĞŶƚĂů ƚĞƐƚƐ ŽŶ ůŝďƌĂƌǇ ƉƌĞƉĂƌĂƚŝŽŶŝŶƚŚĞůĂďŽƌĂƚŽƌǇ͘. 9.

(122) 2.1 Whole genome shotgun assembly ^ĞƋƵĞŶĐŝŶŐŽƌƌĞĂĚŝŶŐƚŚĞĞŶƚŝƌĞŐĞŶŽŵĞŝƐůŝŵŝƚĞĚďǇƚŚĞŵĂǆŝŵƵŵůĞŶŐƚŚŽĨΖĐŽŶƚŝŶƵŽƵƐΖ EƚŚĂƚĐĂŶďĞƐĞƋƵĞŶĐĞĚ͘^ŵĂůůĞƌŐĞŶŽŵĞƐĐŽƵůĚďĞƐĞƋƵĞŶĐĞĚĂƐƐŵĂůůĞƌƉĂƌƚƐĂŶĚƉƵƚ ƚŽŐĞƚŚĞƌ ŝŶƚŽ Ă ŐĞŶĞƚŝĐ ŵĂƉ ďĂƐĞĚ ŽŶ ƚŚĞ ŽǀĞƌůĂƉ ƐŚĂƌĞĚ ďǇ ƚŚĞ ƐĞƋƵĞŶĐĞĚ ƌĞŐŝŽŶƐ͘ &Žƌ ĞǆĂŵƉůĞ͕ ƚŚĞ ͞ƉƐƚĞŝŶͲĂƌƌ sŝƌƵƐ 'ĞŶŽŵĞ͟ ǁĂƐ ĐŽŵƉŝůĞĚ ƚŽŐĞƚŚĞƌ ƵƐŝŶŐ ƉƌĞǀŝŽƵƐůǇ ƉƵďůŝƐŚĞĚĨĞĂƚƵƌĞƐƚŚĂƚŚĂĚďĞĞŶĂŶŶŽƚĂƚĞĚ;ĂĞƌĞƚĂů͕͘ϭϵϴϰͿ͘,ŽǁĞǀĞƌ͕ƐĞƋƵĞŶĐŝŶŐůĂƌŐĞƌ ŐĞŶŽŵĞƐŝƐŵŽƌĞĐŚĂůůĞŶŐŝŶŐ͘dŚĞĨŝƌƐƚůĂƌŐĞƌŐĞŶŽŵĞǁŚĞƌĞƚŚŝƐĐŽƵůĚďĞĂĐĐŽŵƉůŝƐŚĞĚǁĂƐ ƚŚĞŽŶĞŽĨƚŚĞďĂĐƚĞƌŝĂ,ĂĞŵŽƉŚŝůƵƐŝŶĨůƵĞŶǌĂĞ͘ dŚĞ ŐĞŶŽŵĞ ŽĨ ƚŚĞ ďĂĐƚĞƌŝƵŵ ,ĂĞŵŽƉŚŝůƵƐ ŝŶĨůƵĞŶǌĂĞ ǁĂƐ ƐĞƋƵĞŶĐĞĚ ĂŶĚ ĂƐƐĞŵďůĞĚ ďǇ ƌĂŶĚŽŵƐĞƋƵĞŶĐŝŶŐĨŽůůŽǁĞĚďǇĂƐƐĞŵďůǇƚŽŽďƚĂŝŶƚŚĞĨŝƌƐƚĐŽŵƉůĞƚĞŐĞŶŽŵĞƐĞƋƵĞŶĐĞŽĨ Ă ĨƌĞĞ ůŝǀŝŶŐ ŽƌŐĂŶŝƐŵ ;&ůĞŝƐĐŚŵĂŶŶ Ğƚ Ăů͕͘ ϭϵϵϱͿ͘ dŚŝƐ ƐƚƌĂƚĞŐǇ ŽĨ ƐĞƋƵĞŶĐŝŶŐ ǁŚŽůĞ ŐĞŶŽŵĞƐ ďǇ ƌĂŶĚŽŵůǇ ĨƌĂŐŵĞŶƚŝŶŐ E ŝŶƚŽ ƐŵĂůůĞƌ ĨƌĂŐŵĞŶƚƐ ǁŚŝĐŚ ĂƌĞ ƐĞƋƵĞŶĐĞĚ ƐĞƉĂƌĂƚĞůǇ ŝƐ ŬŶŽǁŶ ĂƐ ƐŚŽƚŐƵŶ ƐĞƋƵĞŶĐŝŶŐ͘ /Ŷ ƚŚĞ ǁŚŽůĞ ŐĞŶŽŵĞ ƐŚŽƚŐƵŶ ĂƐƐĞŵďůǇ ƐƚƌĂƚĞŐǇ;&ŝŐƵƌĞϭͿ͕ŐĞŶŽŵŝĐEŝƐƌĂŶĚŽŵůǇďƌŽŬĞŶŝŶƚŽŶƵŵĞƌŽƵƐƐŵĂůůĞƌƉŝĞĐĞƐ͘dŚĞƐĞ ƐŵĂůůĞƌƉŝĞĐĞƐĂƌĞƐĞƋƵĞŶĐĞĚƚŽŽďƚĂŝŶƌĞĂĚƐ͘>ĂƌŐĞŶƵŵďĞƌƐŽĨƐƵĐŚƌĞĂĚƐĂƌĞŐĞŶĞƌĂƚĞĚƐŽ ĂƐ ƚŽ ŽďƚĂŝŶ ŽǀĞƌůĂƉƉŝŶŐ ƌĞĂĚƐ͘ ĂƐĞĚŽŶƚŚĞ ŽǀĞƌůĂƉ ƐŚĂƌĞĚ ďĞƚǁĞĞŶƚŚĞ ĚŝĨĨĞƌĞŶƚ ƌĞĂĚƐ͕ ĐŽŵƉƵƚĞƌ ƉƌŽŐƌĂŵƐ ŬŶŽǁŶ ĂƐ ΖĂƐƐĞŵďůĞƌƐΖ ďƵŝůĚ ƚŚĞƐĞ ƌĞĂĚƐ ŝŶƚŽ Ă ĐŽŶƚŝŶƵŽƵƐ ƐĞƋƵĞŶĐĞ ŬŶŽǁŶĂƐĂΖĐŽŶƚŝŐΖ͘ &ŝŶĚŝŶŐ ƚŚĞ ĐŽƌƌĞĐƚ ŽǀĞƌůĂƉ ďĞƚǁĞĞŶ ŵŝůůŝŽŶƐ ŽĨ ƐŚŽƌƚ ƐĞƋƵĞŶĐĞ ƌĞĂĚƐ ŝƐ Ă ĚŝĨĨŝĐƵůƚ ĂůŐŽƌŝƚŚŵŝĐ ƚĂƐŬ ƚŚĂƚ ŚĂƐ ƐŽ ĨĂƌ ŶŽƚ ĨŽƵŶĚ ĂŶ ĂŶĂůǇƚŝĐĂů ƐŽůƵƚŝŽŶ͘ ,ĞƵƌŝƐƚŝĐ ŵĞƚŚŽĚƐ ǁŚŝĐŚ. 10.

(123) ƵƚŝůŝƐĞ ĞǆƉĞƌŝĞŶĐĞ ďĂƐĞĚ ŵĞƚŚŽĚƐ ĨŽƌ ƐƉĞĞĚŝŶŐ ƵƉ ƐŽůǀŝŶŐ ŽĨ ƉƌŽďůĞŵƐ ĂƌĞ ĐŽŶƚŝŶƵŽƵƐůǇ ďĞŝŶŐ ĚĞǀĞůŽƉĞĚ ;ĂŶŝĞů Ğƚ Ăů͕͘ ϮϬϬϰ͕ ^ĐŚĞŝďǇĞͲůƐŝŶŐ Ğƚ Ăů͕͘ ϮϬϬϵ͕ DŝůůĞƌ Ğƚ Ăů͕͘ ϮϬϭϬͿ ƚŽ ŝŵƉƌŽǀĞƚŚĞƋƵĂůŝƚǇŽĨƚŚĞĂƐƐĞŵďůŝĞƐ͘dŚĞƉƌŽďůĞŵŝƐĞǆĂĐĞƌďĂƚĞĚďǇĞƌƌŽƌƐŝŶƚŚĞƉƌŽĐĞƐƐ ŽĨ ƌĞĂĚŝŶŐ ƚŚĞ ŐĞŶŽŵŝĐ ƐĞƋƵĞŶĐĞ ĂƐ Ă ƌĞƐƵůƚ ŽĨ ůŝŵŝƚĂƚŝŽŶƐ ŝŶ ƚŚĞ ƐĞƋƵĞŶĐŝŶŐ ƚĞĐŚŶŽůŽŐǇ͕ ƉŽůǇŵŽƌƉŚŝƐŵ ŝŶ ŐĞŶŽŵŝĐ E ŽĨ ĚŝƉůŽŝĚ ŽƌŐĂŶŝƐŵƐ͖ ƌĞƉĞĂƚ ƌĞŐŝŽŶƐ ǁŚŝĐŚ ĐĂŶŶŽƚ ďĞ ĚŝƐƚŝŶŐƵŝƐŚĞĚŽƌƉůĂĐĞĚŝŶƚŚĞĐŽƌƌĞĐƚůŽĐĂƚŝŽŶŵĂŬĞƚŚĞƉƌŽĐĞƐƐŽĨŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐĂŶĚ ĂƐƐĞŵďůǇŵŽƌĞĐŽŵƉůŝĐĂƚĞĚŝŶƉƌĂĐƚŝĐĞ͘ . Figure 1: Flowchart of shotgun Genome sequencing . 11.

(124) ǁĂǇƚŽŝŵƉƌŽǀĞƚŚĞĂƐƐĞŵďůǇĂĐƌŽƐƐƌĞƉĞĂƚƌĞŐŝŽŶƐǁŚŝĐŚĐŽŶƐƚŝƚƵƚĞĂƉĂƌƚŝĐƵůĂƌĐŚĂůůĞŶŐĞ ;ĂŶŝĞů Z Ğƚ Ăů͕͘ ϮϬϭϬͿ ĂŶĚ ƚŽ ũŽŝŶ ĐŽŶƚŝŐƐ ǁŝƚŚ ŐĂƉƐ ŝŶ ďĞƚǁĞĞŶ ŝƐ ƚŚĞ ƵƐĞ ŽĨ ĨƌĂŐŵĞŶƚƐ ƐĞƋƵĞŶĐĞĚ ĨƌŽŵ ďŽƚŚ ĞŶĚƐ ǁŝƚŚ ŐĂƉƐ ŽĨ Ă ŬŶŽǁŶ ƐŝǌĞ ŝŶ ďĞƚǁĞĞŶ͘ dŚĞƐĞ ƐƉĞĐŝĂů ƚǇƉĞƐ ŽĨ ƌĞĂĚƐ ĂƌĞ ŐĞŶĞƌĂƚĞĚ ďǇ ƌĂŶĚŽŵůǇ ĨƌĂŐŵĞŶƚŝŶŐ E͕ ƐŝǌĞͲƐĞůĞĐƚŝŶŐ ĂŶĚ ƐĞƋƵĞŶĐŝŶŐ ŝƚ ĨƌŽŵ ďŽƚŚ ĞŶĚƐ ĂŶĚ ĂƌĞ ƵƐƵĂůůǇ ƌĞĨĞƌƌĞĚ ƚŽ ĂƐ ͚ŵĂƚĞ ƉĂŝƌƐΖ Žƌ ũƵŵƉ ůŝďƌĂƌŝĞƐ ;WŽƉ Ğƚ Ăů͕͘ ϮϬϬϰ͕ ^ĂŶƚĞĞƚĂů͕͘ϮϬϭϬͿ͘ΖDĂƚĞƉĂŝƌƐΖƉƌŽǀŝĚĞĂĚĚŝƚŝŽŶĂůŝŶĨŽƌŵĂƚŝŽŶĂďŽƵƚƚŚĞƌĞůĂƚŝǀĞƉŽƐŝƚŝŽŶŽĨ ƚŚĞƉĂŝƌŽĨƌĞĂĚƐǁŚŝĐŚĐĂŶďĞǀĞƌǇƵƐĞĨƵůŝŶĨŽƌŵĂƚŝŽŶŝŶƌĞƐŽůǀŝŶŐƌĞƉĞĂƚƌĞŐŝŽŶƐ͘ŝĨĨĞƌĞŶƚ ƐŝǌĞƐŽĨEĨƌĂŐŵĞŶƚƐ;ŐĞŶĞƌĂůůǇϮ͕ϱ͕ϭϬ͕ϱϬ͕ϭϬϬĂŶĚϭϱϬŬďͿĂƌĞƐĞůĞĐƚĞĚĂŶĚƐĞƋƵĞŶĐĞĚ ĨƌŽŵďŽƚŚĞŶĚƐ͘dŚĞƐĞΖŵĂƚĞƉĂŝƌƐΖŽĨĚŝĨĨĞƌĞŶƚƐŝǌĞƐĂƌĞƵƐĞĚďǇƚŚĞΖĂƐƐĞŵďůĞƌΖƚŽƉŽƐŝƚŝŽŶ ƚŚĞƌĞĂĚƐďĂƐĞĚŽŶĞƐƚŝŵĂƚĞƐŽĨƚŚĞĚŝƐƚĂŶĐĞďĞƚǁĞĞŶƚŚĞƌĞĂĚƉĂŝƌ͘hƐŝŶŐƚŚĞŝŶĨŽƌŵĂƚŝŽŶ ĂǀĂŝůĂďůĞ ŝŶ ƚŚĞ ƌĞĂĚƐ ĂŶĚ ΖŵĂƚĞ ƉĂŝƌƐΖ͕ ƚŚĞ ĐŽŵƉƵƚĞƌ ƉƌŽŐƌĂŵ ǁŝůů ŽƌŐĂŶŝǌĞ ΖĐŽŶƚŝŐƐΖ ŝŶƚŽ ΖƐĐĂĨĨŽůĚƐΖďĂƐĞĚŽŶƚŚĞƌĞůĂƚŝǀĞƉŽƐŝƚŝŽŶŽĨƚŚĞĐŽŶƚŝŐƐďĂƐĞĚŽŶĐŽŶŶĞĐƚŝŽŶƐďĞƚǁĞĞŶŵĂƚĞ ƉĂŝƌƐ͘dŚĞŐĂƉƐƉƌĞƐĞŶƚŝŶƚŚĞƐĐĂĨĨŽůĚƐĂƌĞĨŝůůĞĚďǇĂƉƌŽĐĞƐƐŬŶŽǁŶĂƐŐĞŶŽŵĞĨŝŶŝƐŚŝŶŐ͘ 'ĂƉƐ ƐŵĂůůĞƌ ƚŚĂŶ ϭϬŬď ĂƌĞ ĐŽǀĞƌĞĚ ďǇ WZ ĂŵƉůŝĨŝĐĂƚŝŽŶ ĂŶĚ ƐĞƋƵĞŶĐŝŶŐ ŽĨ ƚŚĞ ƌĞŐŝŽŶ͘ 'ĂƉƐ ďŝŐŐĞƌ ƚŚĂŶ ϭϬŬď ĂƌĞ ŐĞŶĞƌĂůůǇ ĨŝůůĞĚ ƵƐŝŶŐ ŽƚŚĞƌ ƐƚƌĂƚĞŐŝĞƐ ƐƵĐŚ ĂƐ ĐůŽŶĞ ƐĞƋƵĞŶĐŝŶŐ͘ ĐůŽŶĞƐ ƚĂƌŐĞƚ Ă ƐƉĞĐŝĨŝĐ ƌĞĚƵĐĞĚ ƌĞŐŝŽŶ ŽĨ ƚŚĞ ŐĞŶŽŵĞ ƚŚĂƚ ĐĂŶ ďĞ ĂƐƐĞŵďůĞĚǁŝƚŚŽƵƚŚĂǀŝŶŐƚŽƌĞƐŽůǀĞƌĞĂĚƐĨƌŽŵŽƚŚĞƌƌĞŐŝŽŶƐŽĨƚŚĞŐĞŶŽŵĞ͘ 2.1.1 Traditional sanger sequencing dŚĞ ĐŚĂŝŶ ƚĞƌŵŝŶĂƚŝŽŶ ŵĞƚŚŽĚ ;^ĂŶŐĞƌ Ğƚ Ăů͕͘ ϭϵϳϱͿ ŽĨ E ƐĞƋƵĞŶĐŝŶŐ ŚĂƐ ďĞĞŶ ǁŝĚĞůǇ ƵƐĞĚĚƵĞƚŽŝƚƐƌĞůŝĂďŝůŝƚǇĂŶĚƌĞůĂƚŝǀĞĞĂƐĞŽĨƵƐĞ͘/ŶƚŚĞĐŚĂŝŶƚĞƌŵŝŶĂƚŝŽŶŵĞƚŚŽĚƐŝŶŐůĞ ƐƚƌĂŶĚĞĚEƐĂŵƉůĞŝƐĞůŽŶŐĂƚĞĚŝŶĂEƌĞƉůŝĐĂƚŝŽŶƌĞĂĐƚŝŽŶƵƐŝŶŐĂEƉƌŝŵĞƌ͕E ƉŽůǇŵĞƌĂƐĞ͕ĚĞŽǆǇŶƵĐůĞŽƚŝĚĞƉŚŽƐƉŚĂƚĞƐ;ĚEdWƐͿĂŶĚŵŽĚŝĨŝĞĚĚĞŽǆǇŶƵĐůĞŽƚŝĚĞƉŚŽƐƉŚĂƚĞƐ. 12.

(125) ;ĚEdWƐͿǁŚŝĐŚůĂĐŬĂϯΖK,ŐƌŽƵƉ;ƌĞƋƵŝƌĞĚĨŽƌĞƐƚĂďůŝƐŚŝŶŐƉŚŽƐƉŚŽĚŝĞƐƚĞƌďŽŶĚƐďĞƚǁĞĞŶ ĂĚũĂĐĞŶƚŶƵĐůĞŽƚŝĚĞƐͿ͘EĞůŽŶŐĂƚŝŽŶŝƐŝŶƚĞƌƌƵƉƚĞĚǁŚĞŶĂŵŽĚŝĨŝĞĚĚEdWŝƐŝŶĐŽƌƉŽƌĂƚĞĚ͘ ĂĐŚ ŽĨ ƚŚĞ ŵĂŶǇ ŚƵŶĚƌĞĚ ĚŝĨĨĞƌĞŶƚ ƌĞĂĐƚŝŽŶƐ ŝƐ ŝŶƚĞƌƌƵƉƚĞĚ Ăƚ Ă ĚŝĨĨĞƌĞŶƚ ďĂƐĞ ĂůŽŶŐ ƚŚĞ E ƐĞƋƵĞŶĐĞ͘ ZĂĚŝŽĂĐƚŝǀĞůǇ Žƌ ĨůƵŽƌĞƐĐĞŶƚůǇ ůĂďĞůůĞĚ ŵŽĚŝĨŝĞĚ ĚEdWƐ ĐĂŶ ďĞ ǀŝƐƵĂůŝǌĞĚ ĂĨƚĞƌĚĞŶĂƚƵƌŝŶŐŝŶƚŽƐŝŶŐůĞƐƚƌĂŶĚĞĚEĨŽůůŽǁĞĚďǇƐŝǌĞƐĞƉĂƌĂƚŝŽŶ͘sĂƌŝŽƵƐĚEdWΖƐ͕d͕ ' ĂŶĚ ŝƐ ůĂďĞůůĞĚ ǁŝƚŚ Ă ĚŝĨĨĞƌĞŶƚ ĐŽůŽƌĞĚ ĚǇĞ ƚŽ ďĞ ĂďůĞ ƚŽ ŝĚĞŶƚŝĨǇ ďĂƐĞƐ ƉƌĞƐĞŶƚ Ăƚ ĚŝĨĨĞƌĞŶƚƉŽƐŝƚŝŽŶƐĂůŽŶŐƚŚĞƐĞƋƵĞŶĐĞ͘ ZĞĂĚƐ ;ĐŽŶƚŝŶƵŽƵƐ ƐĞƋƵĞŶĐĞ ŽĨ EͿ ŽĨ ĂďŽƵƚ ϭŬď ĐĂŶ ďĞ ƐĞƋƵĞŶĐĞĚ ǁŝƚŚ ƚŚĞ ƚƌĂĚŝƚŝŽŶĂů ^ĂŶŐĞƌƐĞƋƵĞŶĐŝŶŐŵĞƚŚŽĚŽůŽŐǇ͘ĂƚĂŽďƚĂŝŶĞĚĨƌŽŵƚŚŝƐŵĞƚŚŽĚŝƐŽĨƉŽŽƌƋƵĂůŝƚǇĂƚďŽƚŚ ĞŶĚƐŽĨƚŚĞƐĞƋƵĞŶĐĞĚƌĞĂĚ͘,ĞŶĐĞ͕ƋƵĂůŝƚǇǀĂůƵĞƐĂƌĞŐĞŶĞƌĂƚĞĚĨŽƌƚŚĞƐĞƋƵĞŶĐĞĚƌĞĂĚƐƚŽ ĂŝĚ ƚŚĞ ĂƐƐĞŵďůǇ ƉƌŽĐĞƐƐ͘ ĞƉĞŶĚŝŶŐ ŽŶ ƚŚĞ ƐŽĨƚǁĂƌĞ ƵƐĞĚ ĨŽƌ ƉŽƐƚ ƉƌŽĐĞƐƐŝŶŐ ŽĨ ƌĞĂĚƐ ďĂƐĞĚ ŽŶ ƚŚĞƐĞ ƋƵĂůŝƚǇ ƐĐŽƌĞƐ͕ ĞƌƌŽƌ ƌĂƚĞƐ ŽĨ Ϭ͘ϬϬϭ ƚŽ ŵŽƌĞ ƚŚĂŶ ϭй ŚĂǀĞ ďĞĞŶ ƌĞƉŽƌƚĞĚ ;,ŽĨĨϮϬϬϵͿ͘ 2.1.2 NGS: Next-generation sequencing ^ƵďƐĞƋƵĞŶƚ ƚŽ ƐĞƋƵĞŶĐŝŶŐ ŽĨ ƚŚĞ ,ƵŵĂŶ ŐĞŶŽŵĞ͕ ƚŚĞ ŶĞĞĚ ĨŽƌ Ă ĐŽƐƚ ĞĨĨĞĐƚŝǀĞ ĂŶĚ ĨĂƐƚ ŵĞƚŚŽĚŽĨƐĞƋƵĞŶĐŝŶŐŚĂƐůĞĚƚŽƚŚĞĚĞǀĞůŽƉŵĞŶƚŽĨǀĂƌŝŽƵƐŶĞǁƐĞƋƵĞŶĐŝŶŐƚĞĐŚŶŽůŽŐŝĞƐ͘ dŚĞƐĞŶĞǁĞƌŵĞƚŚŽĚƐĂƌĞĐŽůůĞĐƚŝǀĞůǇŬŶŽǁŶĂƐŶĞǆƚͲŐĞŶĞƌĂƚŝŽŶƐĞƋƵĞŶĐŝŶŐ;E'^Ϳ;DŝĐŚĂĞů >͘ D ϮϬϭϬͿ ĂŶĚ ƉƌŽǀŝĚĞ Ă ĐŽƐƚ ĞĨĨĞĐƚŝǀĞ ĂůƚĞƌŶĂƚŝǀĞ ƚŽ ƚƌĂĚŝƚŝŽŶĂů ^ĂŶŐĞƌ ƐĞƋƵĞŶĐŝŶŐ͘ E'^ ŐĞŶĞƌĂƚĞƐ ƐŚŽƌƚĞƌ ƌĞĂĚƐ ŽĨ Ϯϱ ďĂƐĞƉĂŝƌ ƵƉƚŽ ϰϬϬ ďĂƐĞ ƉĂŝƌƐ ŝŶ ůĞŶŐƚŚ ;WĂƵů &ůŝĐĞƌŬ Ğƚ Ăů͕͘ ϮϬϬϵͿĚĞƉĞŶĚŝŶŐŽŶƚŚĞƐĞƋƵĞŶĐŝŶŐƚĞĐŚŶŽůŽŐǇƵƐĞĚ͘. 13.

(126) Sequencing Technology. Cost per Mb of DNA sequence. Cost per human sized Genome. Cost estimated on below Date. Reference. First generation $397.09 platforms (Sanger and other capillary based methods). $7,147,571. October-2007. Wetterstrand KA (Reference 41 ). Second-generation $102.13 platforms (454, SOLID, Illumina, etc). $3,063,820. January-2008. Wetterstrand KA (Reference 41 ). Second-generation $0.23 platforms (454, SOLID, Illumina, etc). $20,963. January-2011. Wetterstrand KA (Reference 41 ). Table 1: Cost comparison of DNA sequencing ƵĞ ƚŽ ƚŚĞ ƐŝŐŶŝĨŝĐĂŶƚ ĐŽƐƚ ďĞŶĞĨŝƚƐ ;ƚĂďůĞ ϭͿ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ E'^ ƚĞĐŚŶŽůŽŐŝĞƐ͕ ƚŚĞǇ ŚĂǀĞ ďĞĐŽŵĞ Ă ƉŽƉƵůĂƌ ƚŽŽů ĨŽƌ ŶŽƚ ŽŶůǇ ŐĞŶŽŵĞ ƌĞͲƐĞƋƵĞŶĐŝŶŐ ďƵƚ ĂůƐŽ ĚĞ ŶŽǀŽ ŐĞŶŽŵĞ ĂƐƐĞŵďůǇ͘ /ƚƐ ƉŽƉƵůĂƌŝƚǇ ǁŝůů ĂƌŐƵĂďůǇ ĞǀĞŶ ŝŶĐƌĞĂƐĞ͕ ƐŝŶĐĞ ƐĞƋƵĞŶĐŝŶŐ ĐŽƐƚƐ ŚĂǀĞ ďĞĞŶ ĚĞĐƌĞĂƐŝŶŐĚƌĂƐƚŝĐĂůůǇƐŝŶĐĞƚŚĞŝŶƚƌŽĚƵĐƚŝŽŶŽĨƚŚĞƐĞĐŽŶĚŐĞŶĞƌĂƚŝŽŶƐĞƋƵĞŶĐŝŶŐŵĞƚŚŽĚƐ ĂŶĚĂƌĞĞǆƉĞĐƚĞĚƚŽĐŽŶƚŝŶƵĞƚŽĚŽƐŽĨŽƌĂǁŚŝůĞ͘ 2.1.3 Genome assembly and its challenges. ĚǀĂŶĐĞƐ ŝŶ ƐĞƋƵĞŶĐŝŶŐ ƚĞĐŚŶŽůŽŐǇ ŚĂǀĞ ďĞĞŶ ƌĞĨůĞĐƚĞĚ ŝŶ ƚŚĞ ŵĞƚŚŽĚƐ ĨŽƌ ĂƐƐĞŵďůŝŶŐ ŐĞŶŽŵĞƐ͘ ĞĨŽƌĞ ƚŚĞ ĂĚǀĞŶƚ ŽĨ E'^ ƚĞĐŚŶŽůŽŐŝĞƐ ĂƐƐĞŵďůǇ ƉƌŽŐƌĂŵƐ ƌĞůŝĞĚ ŽŶ ͞ŽǀĞƌůĂƉͲ ůĂǇŽƵƚͲĐŽŶƐĞŶƐƵƐ͟ŵĞƚŚŽĚƐĨŽƌŐĞŶŽŵĞĂƐƐĞŵďůǇ͘>ŽŶŐƌĞĂĚůĞŶŐƚŚƐĂŶĚĂĞƌƌŽƌƌĂƚĞŽĨϭй ŵĂĚĞ ŝƚ ƉŽƐƐŝďůĞ ƚŽ ĂƐƐĞŵďůĞ ŐĞŶŽŵĞƐ ďĂƐĞĚ ŽŶ ŽǀĞƌůĂƉ ŐƌĂƉŚƐ ;ĞŶŝƐŽǀ Ğƚ Ăů͕͘ ϮϬϬϴͿ͘. 14.

(127) ^ŚŽƌƚĞƌƌĞĂĚůĞŶŐƚŚƐ͕ĞǆƉŽŶĞŶƚŝĂůŝŶĐƌĞĂƐĞŝŶƚŚĞŶƵŵďĞƌŽĨƌĞĂĚƐĂŶĚŚŝŐŚĞƌĞƌƌŽƌƌĂƚĞƐĂƌĞ ŶŽƚƐƵŝƚĂďůĞĨŽƌƚŚĞƐĞĂůŐŽƌŝƚŚŵƐ͘,ĞŶĐĞ͕ǀĂƌŝŽƵƐĂƐƐĞŵďůǇƉƌŽŐƌĂŵƐ;ĂŶŝĞůZĞƚĂů͕͘ϮϬϭϬͿ ďĂƐĞĚŽŶĚĞƌƵŝũŶ'ƌĂƉŚƐŚĂǀĞďĞĞŶĐƌĞĂƚĞĚ͘ĚĞƌƵŝũŶŐƌĂƉŚƐŝƐĂƚǇƉĞŽĨĚŝƌĞĐƚĞĚŐƌĂƉŚ ƵƐĞĚƚŽƌĞƉƌĞƐĞŶƚŽǀĞƌůĂƉƐďĞƚǁĞĞŶƐĞƋƵĞŶĐĞƐŽĨƐǇŵďŽůƐ͘ dŚĞƐĞ ĂƉƉƌŽĂĐŚĞƐ ƚƌĞĂƚ ƚŚĞ ĚĂƚĂ ĂƐ ǁŽƌĚƐ ŽĨ Ŭ ŶƵŵďĞƌ ŽĨ ŶƵĐůĞŽƚŝĚĞ ďĂƐĞƐ Žƌ ŬͲŵĞƌƐ ŝŶƐƚĞĂĚŽĨƌĞĂĚƐ͘,ŝŐŚĞƌƌĞĚƵŶĚĂŶĐǇŐĞŶĞƌĂƚĞĚďǇE'^ŵĞƚŚŽĚƐŝƐŚĂŶĚůĞĚďǇƚŚŝƐŵĞƚŚŽĚ ĂƐƌĞĂĚƐĂƌĞďƌŽŬĞŶŝŶƚŽΖŬͲŵĞƌƐ͛ƚŚĂƚŵĂŬĞƵƉĞĂĐŚƌĞĂĚ͘dŚĞĚĞƌƵŝũŶŐƌĂƉŚŝƐĐŽŶƐƚƌƵĐƚĞĚ ďǇƌĞƉƌĞƐĞŶƚŝŶŐĂƐĞƌŝĞƐŽĨŽǀĞƌůĂƉƉŝŶŐŬͲŵĞƌƐĂƐĂŶŽĚĞŝŶƚŚĞŐƌĂƉŚ͘ĨƚĞƌĐŽŶƐƚƌƵĐƚŝŽŶŽĨ ƚŚĞ ŐƌĂƉŚ ĂŶĚ ΖŚĂƐŚŝŶŐΖ ƚŚĞ ƌĞĂĚƐ ďĂƐĞĚ ŽŶ ŬͲŵĞƌΖƐ ƚŚĂƚ ŵĂŬĞ ƵƉ ƚŚĞ ƌĞĂĚ͕ ƚŚĞ ŐƌĂƉŚ ŝƐ ƐŝŵƉůŝĨŝĞĚ ǁŝƚŚŽƵƚ ůŽƐŝŶŐ ĚĂƚĂ ƵƐŝŶŐ ƐƚƌŝŶŐ ŐƌĂƉŚ ďĂƐĞĚ ŵĞƚŚŽĚƐ͘ ƐƚƌŝŶŐ ŐƌĂƉŚ ŝƐ ĂŶ ŝŶƚĞƌƐĞĐƚŝŽŶŐƌĂƉŚŽĨĐƵƌǀĞƐŝŶƚŚĞƉůĂŶĞ͕ǁŚĞƌĞĞĂĐŚĐƵƌǀĞŝƐĐĂůůĞĚĂƐƚƌŝŶŐ͘ZĞƉƌĞƐĞŶƚŝŶŐ ƚŚĞ ŝŶƚĞƌƐĞĐƚŝŽŶ ŽĨ ƚŚĞ ĚŝĨĨĞƌĞŶƚ ŬͲŵĞƌ͛Ɛ ĂůůŽǁƐ ƚŚĞ ƵƐĞ ŽĨ ƐƚƌŝŶŐ ŐƌĂƉŚ ƐŝŵƉůŝĨŝĐĂƚŝŽŶ ŵĞƚŚŽĚƐ͘ ^ŝŵƉůŝĨŝĐĂƚŝŽŶ ŽĨ ƚŚĞ ŐƌĂƉŚ ĞǆƉĞĚŝƚĞƐ ƚŚĞ ƐƵďƐĞƋƵĞŶƚ ĞƌƌŽƌ ƌĞŵŽǀĂů͕ ƌĞƉĞĂƚ ƌĞƐŽůƵƚŝŽŶ ĂŶĚ ƐĐĂĨĨŽůĚŝŶŐ ƐƚĞƉƐ͘ /ŵƉƌŽǀĞŵĞŶƚƐ ŝŶ ĞƌƌŽƌ ŚĂŶĚůŝŶŐ ĂŶĚ ƌĞƉĞĂƚ ƌĞƐŽůƵƚŝŽŶ ĂůŐŽƌŝƚŚŵƐ ŚĂǀĞ ŵĂĚĞ ŝƚ ƉŽƐƐŝďůĞ ƚŽ ĂƐƐĞŵďůĞ ǁŚŽůĞ ŐĞŶŽŵĞƐ ƵƐŝŶŐ ũƵƐƚ ƐŚŽƌƚͲƌĞĂĚ ƐĞƋƵĞŶĐŝŶŐ;ZƵŝƋŝĂŶŐ>ŝĞƚĂů͕͘ϮϬϭϬͿ͘dŚĞŽǀĞƌůĂƉĐŽŶƐĞŶƐƵƐďĂƐĞĚĂƉƉƌŽĂĐŚ͕ŽŶƚŚĞŽƚŚĞƌ ŚĂŶĚ͕ ƌĞůŝĞƐ ĞŶƚŝƌĞůǇ ŽŶ ĨŝŶĚŝŶŐ ŽǀĞƌůĂƉƐ ďĞƚǁĞĞŶ ƌĞĂĚƐ ĨŽƌ ƉĞƌĨŽƌŵŝŶŐ ƚŚĞ ĂƐƐĞŵďůǇ͘ WƌĞƐĞŶĐĞ ŽĨ ƌĞĚƵŶĚĂŶƚ ŝŶĨŽƌŵĂƚŝŽŶ ŵĂŬĞƐ ƚŚĞ ŽǀĞƌůĂƉ ďĂƐĞĚ ŵĞƚŚŽĚ ĐƵŵďĞƌƐŽŵĞ ĂƐ ŵĞŵŽƌǇƌĞƋƵŝƌĞŵĞŶƚƐĂƌĞǀĞƌǇŚŝŐŚ͘ . 15.

(128) ĞƐƉŝƚĞ ŽĨ ƚŚĞ ĚŝĨĨŝĐƵůƚŝĞƐ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ƐŚŽƌƚ ƌĞĂĚ ĚĂƚĂ͕ ƐĞƋƵĞŶĐŝŶŐ ŽĨ 'ŝŐĂ ďĂƐĞ ƐŝǌĞĚ ŐĞŶŽŵĞƐ ƵƐŝŶŐ ƐŚŽƌƚͲƌĞĂĚ ƐĞƋƵĞŶĐŝŶŐ ŚĂƐ ďĞĞŶ ĚĞŵŽŶƐƚƌĂƚĞĚ ǁŝƚŚ ĐŽŶƐŝĚĞƌĂďůĞ ƐƵĐĐĞƐƐ ;ZƵŝƋŝĂŶŐ>ŝĞƚĂů͕͘ϮϬϭϬ͕^ĐŚĂƚǌĞƚĂů͕͘ϮϬϭϬͿ͘ dŚĞŚŝŐŚĞƌĞƌƌŽƌƌĂƚĞŝŶƚŚĞƐŚŽƌƚͲƌĞĂĚƐĞƋƵĞŶĐŝŶŐĂůŽŶŐǁŝƚŚƚŚĞƉŽůǇŵŽƌƉŚŝƐŵƉƌĞƐĞŶƚŝŶ ƚŚĞŐĞŶŽŵĞŝŵƉŽƐĞƐĂůŝŵŝƚŽŶƚŚĞŵĂǆŝŵƵŵĐŽŶƚŝŐƵŝƚǇƚŚĂƚĐĂŶďĞĂƐƐĞŵďůĞĚ͘/ƚŚĂƐďĞĞŶ ƐŚŽǁŶ ƚŚĂƚ ƚŚĞ ĐŽŶƚŝŐƵŝƚǇ ŵĞĂƐƵƌĞĚŝŶƚĞƌŵƐŽĨ EϱϬ ;ƚŚĞ ůĞŶŐƚŚ y ŽĨƚŚĞ ƐĞƋƵĞŶĐĞ ǁŚŝĐŚ ŚĂƐ ϱϬй ŽĨ Ăůů ďĂƐĞƐ ŝŶ ƐĞƋƵĞŶĐĞƐ ůŽŶŐĞƌ ƚŚĂŶ yͿ ŽĨ ƚŚĞ ĂƐƐĞŵďůĞĚ ŐĞŶŽŵĞ ƌĞĂĐŚĞƐ Ă ŵĂǆŝŵƵŵǀĂůƵĞĂƚĂĐŽǀĞƌĂŐĞŽĨĂƌŽƵŶĚϱϬyĂŶĚĂĐƚƵĂůůǇĚĞĐƌĞĂƐĞƐĂƚŚŝŐŚĞƌĐŽǀĞƌĂŐĞǁŝƚŚ ϭй ƐĞƋƵĞŶĐŝŶŐ ĞƌƌŽƌ ĂŶĚ ϭй ^EWƐ ŝŶ ƚŚĞ ƌĂǁ ĚĂƚĂ ƵƐĞĚ ĨŽƌ ĂƐƐĞŵďůǇ ;ĂŶŝĞů ϮϬϬϵͿ͘ ĞƉĞŶĚŝŶŐ ŽŶ ƚŚĞĚĂƚĂƐĞƚ ƵƐĞĚƚŚĞ ŵĂǆŝŵƵŵ ĐŽŶƚŝŐƵŝƚǇ ŝƐ ŽďƚĂŝŶĞĚ ďĞƚǁĞĞŶ ĐŽǀĞƌĂŐĞ ŽĨ ϰϬĂŶĚϲϬy͘ dƌĂĚŝƚŝŽŶĂůůǇ ƌĞƉĞĂƚ ƌĞŐŝŽŶƐ ŚĂǀĞ ďĞĞŶ ƌĞƐŽůǀĞĚ ďǇ ƐĐĂĨĨŽůĚŝŶŐ ƵƐŝŶŐ ŵĂƚĞ ƉĂŝƌƐ ŽĨ ǀĂƌŝŽƵƐ ŝŶƐĞƌƚ ƐŝǌĞ͘ ŝĨĨĞƌĞŶƚ ŝŶƐĞƌƚ ƐŝǌĞ ůŝďƌĂƌŝĞƐ ŚĂǀĞ ďĞĞŶ ƵƐĞĚ ǁŝƚŚ ǀĂƌǇŝŶŐ ĚĞŐƌĞĞƐ ŽĨ ƵƚŝůŝƚǇ͘ ŽŶƚŝŐƵŝƚǇ͕ ĐŽƐƚ ďĞŶĞĨŝƚƐ͕ ĂĐĐƵƌĂĐǇ͕ ŐĞŶŽŵĞ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ŽďƚĂŝŶĞĚ ďǇ ƚŚĞ ĚŝĨĨĞƌĞŶƚ ŵĞƚŚŽĚƐŚĂƐƐŚŽǁŶƚŚĂƚĞĂĐŚŵĞƚŚŽĚŚĂƐŝƚƐŽǁŶĂĚǀĂŶƚĂŐĞƐĂŶĚĚŝƐĂĚǀĂŶƚĂŐĞƐ;>ŝĂŶŐzĞ Ğƚ Ăů͕͘ ϮϬϭϭͿ͘ ZĞƉĞĂƚ ĐŽŶƚĞŶƚ ĂŶĚ ' ĐŽŶƚĞŶƚ ŽĨ ŐĞŶŽŵĞƐ ďĞŝŶŐ ƐĞƋƵĞŶĐĞĚ ĂůƐŽ ŚĂǀĞ Ă ĐŽŶƐŝĚĞƌĂďůĞŝŵƉĂĐƚŽŶůŝďƌĂƌǇƉƌĞƉĂƌĂƚŝŽŶ͕ƐĞƋƵĞŶĐŝŶŐĂŶĚĂƐƐĞŵďůǇŵĞƚŚŽĚƐ͘ ZĞĐĞŶƚůǇ͕ ŚŝŐŚ ƋƵĂůŝƚǇ ŐĞŶŽŵĞ ĂƐƐĞŵďůŝĞƐ ŽĨ ŵĂŵŵĂůŝĂŶ ŐĞŶŽŵĞƐ ǁĞƌĞ ĚŽŶĞ ĨƌŽŵ ŵĂƐƐŝǀĞůǇ ƉĂƌĂůůĞů ƐĞƋƵĞŶĐĞ ĚĂƚĂ ;^ĂŶƚĞ Ğƚ Ăů͕͘ ϮϬϭϬͿ ǁŝƚŚ Ă ƋƵĂůŝƚǇ ǀĞƌǇ ƐŝŵŝůĂƌ ƚŽ ƚŚĂƚ. 16.

(129) ŽďƚĂŝŶĞĚ ĨƌŽŵ ^ĂŶŐĞƌ ƐĞƋƵĞŶĐŝŶŐ͘ hƐŝŶŐ ƐƉĞĐŝĨŝĐ ƚǇƉĞƐ ŽĨ ůŝďƌĂƌŝĞƐ ĂŶĚ ŶĞǁ ĂƐƐĞŵďůǇ ƉƌŽŐƌĂŵƐ;>>Wd,^Ͳ>'ͿŚŝŐŚƋƵĂůŝƚǇĂƐƐĞŵďůŝĞƐŚĂǀĞďĞĞŶŽďƚĂŝŶĞĚ͘ 2.2 Partitioning the genome to reduce the problem of assembly 2.2.1 BAC clones KŶĞǁĂǇƚŽƌĞĚƵĐĞƚŚĞƉƌŽďůĞŵŽĨĂƐƐĞŵďůǇŝƐƚŽƌĞĚƵĐĞƚŚĞĂƐƐĞŵďůǇƚĂƌŐĞƚƐŝǌĞ͘'ĞŶŽŵŝĐ ůŝďƌĂƌŝĞƐ ŚĂǀĞ ďĞĞŶ ĚŝǀŝĚĞĚ ŝŶƚŽ ƐŵĂůůĞƌ ƉĂƌƚƐ ƚŚƌŽƵŐŚ ƚŚĞ ƵƐĞ ŽĨ ĂĐƚĞƌŝĂů ĂƌƚŝĨŝĐŝĂů ĐŚƌŽŵŽƐŽŵĞƐ ;ΖƐͿ Žƌ zĞĂƐƚ ĂƌƚŝĨŝĐŝĂů ĐŚƌŽŵŽƐŽŵĞƐ ;zƐͿ͘ Ɛ ĂƌĞ ĐŽŶƐƚƌƵĐƚĞĚ ďǇ ƉĂƌƚŝĂůůǇĚŝŐĞƐƚŝŶŐĂŶĚĨƌĂŐŵĞŶƚŝŶŐŐĞŶŽŵŝĐEƚŽŽďƚĂŝŶůĂƌŐĞĐŚƵŶŬƐŽĨƚŚĞŐĞŶŽŵĞƚŚĂƚ ĂƌĞƚŚĞŶƉƌĞƐĞƌǀĞĚŝŶďĂĐƚĞƌŝĂůĐŽůŽŶŝĞƐ͘dƌĂĚŝƚŝŽŶĂůůǇůŝďƌĂƌŝĞƐŚĂǀĞďĞĞŶĐŽŶƐƚƌƵĐƚĞĚ ďĂƐĞĚŽŶƚŚĞ͞ĚŝǀŝĚĞĂŶĚĐŽŶƋƵĞƌ͟ƐƚƌĂƚĞŐǇƚŽŽďƚĂŝŶŚŝŐŚƋƵĂůŝƚǇŐĞŶŽŵĞĂƐƐĞŵďůŝĞƐƵƐŝŶŐ ^ĂŶŐĞƌƐĞƋƵĞŶĐŝŶŐ͘WĂƌƚŝƚŝŽŶŝŶŐƚŚĞŐĞŶŽŵĞŝŶƚŽƐŵĂůůĞƌĨƌĂŐŵĞŶƚƐƚŚĂƚĐĂŶďĞĂƐƐĞŵďůĞĚ ƐĞƉĂƌĂƚĞůǇƐŝŵƉůŝĨŝĞƐƚŚĞĂƐƐĞŵďůǇƉƌŽďůĞŵĂŶĚƉƌŽǀŝĚĞƐŵŽƌĞĐŽŶƚŝŐƵŽƵƐĂƐƐĞŵďůŝĞƐ͘/ƚŚĂƐ ďĞĞŶ ĨŽƵŶĚ ƚŚĂƚ ďŽƚŚ ůŽŶŐ ĂŶĚ ƐŚŽƌƚ ƌĂŶŐĞ ŵŝƐͲĂƐƐĞŵďůŝĞƐ ĐĂŶ ďĞ ƌĞĚƵĐĞĚ ŝŶ ďĂƐĞĚ ĂƉƉƌŽĂĐŚĞƐĚƵĞƚŽƐĞƋƵĞŶĐŝŶŐŝŶĨŽƌŵĂƚŝŽŶďĞŝŶŐƌĞƐƚƌŝĐƚĞĚƚŽƚŚĞŐĞŶŽŵŝĐƌĞŐŝŽŶĐŽǀĞƌĞĚ ďǇƚŚĞĐůŽŶĞ;DĂƌƌĂĞƚĂů͕͘ϭϵϵϴͿ͘dŚĞŐĞŶŽŵĞĐĂŶďĞƐƉůŝƚŝŶƚŽĐůŽŶĞƐĂŶĚĚŝĨĨĞƌĞŶƚ ƉĂƌƚƐĐĂŶďĞƐĞƋƵĞŶĐĞĚďǇĚŝĨĨĞƌĞŶƚŐƌŽƵƉƐƐŝŵƵůƚĂŶĞŽƵƐůǇĂƐǁĂƐĚŽŶĞǁŚŝůĞƐĞƋƵĞŶĐŝŶŐ ƚŚĞŚƵŵĂŶŐĞŶŽŵĞ͘ ůŝďƌĂƌŝĞƐŚĂǀĞďĞĞŶƉŽŽůĞĚĂŶĚƐĞƋƵĞŶĐĞĚƵƐŝŶŐE'^ƚĞĐŚŶŽůŽŐŝĞƐƚŽŽďƚĂŝŶĂƐƐĞŵďůŝĞƐ ƚŚĂƚďƌŝŶŐƚŽŐĞƚŚĞƌĂŶĚE'^ŵĞƚŚŽĚŽůŽŐŝĞƐ͘/ƚŚĂƐĂůƐŽďĞĞŶƐŚŽǁŶ;EŝŝŶĂĞƚĂů͕͘ϮϬϭϭͿ ƚŚĂƚ ƉŽŽůĞĚ ƐƚƌĂƚĞŐǇ ĨŽƌ ǁŚŽůĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐŝŶŐ ŚĂƐ ŵĂŶǇ ďĞŶĞĨŝƚƐ ƐƵĐŚ ĂƐ ůŽǁĞƌ. 17.

(130) ĐŽƐƚ ĂŶĚ ďĞƚƚĞƌ ĂƐƐĞŵďůǇ ƋƵĂůŝƚǇ ĐŽŵƉĂƌĞĚ ƚŽ ǁŚŽůĞ ŐĞŶŽŵĞ ƐŚŽƚŐƵŶ ƐĞƋƵĞŶĐŝŶŐ͘ ,ŽǁĞǀĞƌ͕ ůŝďƌĂƌŝĞƐ ĂƌĞ ĞǆƉĞŶƐŝǀĞ ƚŽ ĐŽŶƐƚƌƵĐƚ ĂŶĚ ŵĂŝŶƚĂŝŶ͘ ůŝďƌĂƌŝĞƐ ƐƵĨĨĞƌ ĨƌŽŵ ƐŽŵĞĨŽƌŵŽĨǀĂƌŝĂďůĞĐůŽŶŝŶŐďŝĂƐǁŚŝĐŚůĞĂĚƐƚŽŽǀĞƌƌĞƉƌĞƐĞŶƚĂƚŝŽŶŽĨƐŽŵĞƌĞŐŝŽŶƐƚŚĂƚ ĂƌĞĐůŽŶĞĚďĞƚƚĞƌĂƚƚŚĞĞǆƉĞŶƐĞŽĨŽƚŚĞƌƌĞŐŝŽŶƐƚŚĂƚĂƌĞŶŽƚĐůŽŶĞĚ͘^ŝŶĐĞ͕ƚŚĞĂŶĚ z ƐƚƌĂƚĞŐŝĞƐ ŝŶǀŽůǀĞ ƌĂŶĚŽŵ ĨƌĂŐŵĞŶƚĂƚŝŽŶ͖ ƚŚĞ ƐĂŵĞ ůŝďƌĂƌŝĞƐ ĐĂŶŶŽƚ ďĞ ƌĞƉƌŽĚƵĐĞĚ͘ ŽŶƚĂŵŝŶĂƚŝŽŶĨƌŽŵǀĂƌŝŽƵƐƐŽƵƌĐĞƐƐƵĐŚĂƐƚŚĞĐůŽŶŝŶŐǀĞĐƚŽƌƵƐĞĚƚŽƉƌŽĚƵĐĞƚŚĞĐůŽŶĞ ĐĂŶĂůƐŽďĞĂŵĂũŽƌƉƌŽďůĞŵǁŚŝůĞƵƐŝŶŐďĂĐƚĞƌŝĂŽƌǇĞĂƐƚƐ͘ 2.2.2 Reduced representation library based genome assembly ŽŶƐƚƌƵĐƚŝŽŶ ŽĨ Ă ƐĞƌŝĞƐ ŽĨ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ůŝďƌĂƌŝĞƐ ďǇ ƐŝǌĞ ĨƌĂĐƚŝŽŶĂƚŝŽŶ ŚĂƐ ďĞĞŶ ƐĞĞŶ ĂƐ ĂŶŽƚŚĞƌ ƉŽƐƐŝďůĞ ǁĂǇ ĨŽƌ ƉĂƌƚŝƚŝŽŶŝŶŐ ƚŚĞ ŐĞŶŽŵĞ͘ dŚĞ ŐĞŶŽŵĞ ŝƐ ĨƌĂŐŵĞŶƚĞĚ ďǇ ĚŝŐĞƐƚŝŶŐ ǁŝƚŚ Ă ƌĞƐƚƌŝĐƚŝŽŶ ĞŶǌǇŵĞ ƚŽ ŽďƚĂŝŶ Ă ƌĞƉƌŽĚƵĐŝďůĞ ƐĞƚ ŽĨ ĨƌĂŐŵĞŶƚƐ͘ dŚĞƐĞ ĨƌĂŐŵĞŶƚƐ ĂƌĞ ƐĞƉĂƌĂƚĞĚ ďĂƐĞĚ ŽŶ ƐŝǌĞ ŝŶƚŽ ĚŝƐƚŝŶĐƚ ůŝďƌĂƌŝĞƐ ďĂƐĞĚ ŽŶ ƐŝǌĞ ƚŽ ŽďƚĂŝŶ ƌĞƉƌŽĚƵĐŝďůĞŐĞŶŽŵŝĐƉĂƌƚŝƚŝŽŶƐ͘ tŚŽůĞ ŐĞŶŽŵĞ ĂƐƐĞŵďůǇ ƵƐŝŶŐ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ůŝďƌĂƌŝĞƐ ĂŶĚ ƐŚŽƌƚ ƌĞĂĚƐ ŚĂƐ ďĞĞŶ ĚĞŵŽŶƐƚƌĂƚĞĚ ƚŽ ďĞ ĞĨĨĞĐƚŝǀĞ ;zŽƵŶŐ Ğƚ Ăů͕͘ ϮϬϭϬͿ ǁŝƚŚ ƚŚĞ ƌŽƐŽƉŚŝůĂ ŐĞŶŽŵĞ ĂŶĚ ŝŶ ĐŽŵƉĂƌŝƐŽŶ ǁŝƚŚ Ă ƌĞĨĞƌĞŶĐĞ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐĞĚ ďǇ ƚƌĂĚŝƚŝŽŶĂů ^ĂŶŐĞƌ ƐĞƋƵĞŶĐŝŶŐ͘ ĞƚƚĞƌ ƋƵĂůŝƚǇ ĂƐƐĞŵďůŝĞƐ ǁĞƌĞ ŽďƚĂŝŶĞĚ ƵƐŝŶŐ ƌĞƐƚƌŝĐƚŝŽŶ ĞŶǌǇŵĞƐ ƚŽ ĐƌĞĂƚĞ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶƚŚĂŶƵƐŝŶŐǁŚŽůĞŐĞŶŽŵĞůŝďƌĂƌǇĂƐƐĞŵďůŝĞƐ͘ . 18.

(131) ZĞƐƚƌŝĐƚŝŽŶ ĞŶǌǇŵĞ ďĂƐĞĚ ĨƌĂŐŵĞŶƚĂƚŝŽŶ ŽĨ ŐĞŶŽŵĞƐ ƉƌŽĚƵĐĞƐ ƌĞƉƌŽĚƵĐŝďůĞ ĨƌĂŐŵĞŶƚƐ͘ ,ĞŶĐĞ͕ƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌŝĞƐŚĂǀĞďĞĞŶƵƐĞĚĨŽƌŽďƚĂŝŶŝŶŐ^EWŵĂƉƐ;ůƚƐŚƵůĞƌ ĞƚĂů͕͘ϮϬϬϬͿ͘/ƚŚĂƐďĞĞŶƐƵŐŐĞƐƚĞĚ;zŽƵŶŐĞƚĂů͕͘ϮϬϭϬͿƚŚĂƚƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌŝĞƐ ĂƌĞĞĂƐŝĞƌĂŶĚĐŚĞĂƉĞƌƚŽƉƌŽĚƵĐĞƚŚĂŶŽƌzĐůŽŶĞƐ͘ ZĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ůŝďƌĂƌŝĞƐ ĐĂŶ ďĞ ƉƌŽĚƵĐĞĚ ďǇ ĚŝŐĞƐƚŝŶŐ ŐĞŶŽŵŝĐ E ǁŝƚŚ Ă ƌĞƐƚƌŝĐƚŝŽŶĞŶǌǇŵĞĨŽůůŽǁĞĚďǇĞůĞĐƚƌŽƉŚŽƌĞƚŝĐƐŝǌĞƐĞƉĂƌĂƚŝŽŶŽĨŐĞŶŽŵŝĐE͘EĨƌŽŵ ĚŝĨĨĞƌĞŶƚ ƐŝǌĞ ƌĂŶŐĞ ĂƌĞ ĐƵƚ ŽƵƚ ĨƌŽŵ ƚŚĞ ŐĞů ĂŶĚ ƉƵƌŝĨŝĞĚ͘ ĂĐŚ ůŝďƌĂƌǇ ƚŚĞŶ ĐŽŶƐŝƐƚƐ ŽĨ ĨƌĂŐŵĞŶƚƐ ŽĨ Ă ƉĂƌƚŝĐƵůĂƌ ƐŝǌĞ ƌĂŶŐĞ͘ /ŶĚŝǀŝĚƵĂů ůŝďƌĂƌŝĞƐ ĂƌĞ ƐĞƋƵĞŶĐĞĚ ƐĞƉĂƌĂƚĞůǇ Žƌ ƐĞƋƵĞŶĐĞĚƚŽŐĞƚŚĞƌĂĨƚĞƌƚĂŐŐŝŶŐƚŚĞŵǁŝƚŚĂƵŶŝƋƵĞƐĞƋƵĞŶĐĞŵĂƌŬĞƌďǇĂƉƌŽĐĞƐƐŬŶŽǁŶ ĂƐďĂƌĐŽĚŝŶŐ͘ƐƐĞŵďůŝŶŐƚŚĞƐĞůŝďƌĂƌŝĞƐƐĞƉĂƌĂƚĞůǇŚĂƐďĞŶĞĨŝƚƐƐŝŵŝůĂƌƚŽƚŚŽƐĞŽďƚĂŝŶĞĚ ďǇƵƐŝŶŐůŝďƌĂƌŝĞƐ͘ /ƚ ŝƐ ĞǆƉĞĐƚĞĚ ƚŚĂƚ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ůŝďƌĂƌŝĞƐ ǁŝůů ƌĞĚƵĐĞ ŵŝƐͲĂƐƐĞŵďůŝĞƐ ǁŝƚŚŽƵƚ ŝŶƚƌŽĚƵĐŝŶŐ ƉƌŽďůĞŵƐ ĂƐƐŽĐŝĂƚĞĚ ǁŝƚŚ ƚŚĞ ĂƉƉƌŽĂĐŚ͘ ƉĂƌƚ ĨƌŽŵ ďĞŝŶŐ ĞǆƉĞŶƐŝǀĞ ƚŽ ĐŽŶƐƚƌƵĐƚĂŶĚŵĂŝŶƚĂŝŶ͕ůŝďƌĂƌŝĞƐĂƌĞƉƌŽŶĞƚŽĐŽŶƚĂŵŝŶĂƚŝŽŶĂŶĚǀĂƌŝĂďůĞĐůŽŶŝŶŐďŝĂƐ ǁŚŝĐŚůĞĂĚƐƚŽŽǀĞƌƌĞƉƌĞƐĞŶƚĂƚŝŽŶŽĨƐŽŵĞƌĞŐŝŽŶƐĂƚƚŚĞĞǆƉĞŶƐĞŽĨŽƚŚĞƌƌĞŐŝŽŶƐ͘hƐŝŶŐ ƐŝŶŐůĞ ĞŶĚ ƐĞƋƵĞŶĐŝŶŐ ĨŽƌ ƌŽƐŽƉŚŝůĂ ŐĞŶŽŵĞ͕ zŽƵŶŐ Ğƚ Ăů͕͘ ŚĂǀĞ ƐŚŽǁŶ ƚŚĂƚ ƌĞĂĚƐ ŐĞŶĞƌĂƚĞĚ ďǇ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ĂƉƉƌŽĂĐŚ ŐŝǀĞƐ Ă ďĞƚƚĞƌ ĂƐƐĞŵďůǇ ǁŚĞŶ ĞĂĐŚ ŽĨ ƚŚĞ ůŝďƌĂƌŝĞƐŝƐĂƐƐĞŵďůĞĚƐĞƉĂƌĂƚĞůǇĂŶĚƉƵƚƚŽŐĞƚŚĞƌŚŝĞƌĂƌĐŚŝĐĂůůǇƌĂƚŚĞƌƚŚĂŶĂƐƐĞŵďůŝŶŐĂůů ƚŚĞƌĞĂĚƐƚŽŐĞƚŚĞƌ͘,ŽǁĞǀĞƌ͕ƌĞĂĚƐŐĞŶĞƌĂƚĞĚďǇƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶĂƉƉƌŽĂĐŚǁŝůůůĂĐŬ ŽǀĞƌůĂƉƉŝŶŐƌĞĂĚƐĂƚƌĞƐƚƌŝĐƚŝŽŶĞŶǌǇŵĞĐƵƚƐŝƚĞƐǁŚŝĐŚĐŽƵůĚůĞĂĚƚŽƉŽŽƌƉĞƌĨŽƌŵĂŶĐĞŽĨ. 19.

(132) ƚŚĞǁŚŽůĞŐĞŶŽŵĞĂƐƐĞŵďůǇŵĞƚŚŽĚ͘,ĞŶĐĞ͕ǁĞƐŝŵƵůĂƚĞƌĞĂĚƐĨŽƌƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ĂƉƉƌŽĂĐŚĂŶĚǁŚŽůĞŐĞŶŽŵĞĂƉƉƌŽĂĐŚƐĞƉĂƌĂƚĞůǇ͘ 2.3 Sequencing the crow genome. /Ŷ ƚŚŝƐ ƉƌŽũĞĐƚ ǁĞ ƚƌŝĞĚ ƚŽ ĂƐĐĞƌƚĂŝŶ ƚŚĞ ĨĞĂƐŝďŝůŝƚǇ ĂŶĚ ƵƚŝůŝƚǇ ŽĨ ĂƉƉůǇŝŶŐ ƐƵĐŚ Ă ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌǇďĂƐĞĚĂƉƉƌŽĂĐŚƚŽŐĞŶŽŵĞĂƐƐĞŵďůǇŝŶĂůĂƌŐĞĂǀŝĂŶŐĞŶŽŵĞŽĨŽŶĞ ŚŽŽĚĞĚĐƌŽǁ;ŽƌǀƵƐĐŽƌŶŝǆͿŝŶĚŝǀŝĚƵĂů͘dŚĞŵŽƚŝǀĂƚŝŽŶĨŽƌŐĞŶŽŵĞƐĞƋƵĞŶĐŝŶŐŽĨƚŚĞĐƌŽǁ ĐŽŵĞƐĨƌŽŵŝƚƐŝŵƉŽƌƚĂŶĐĞŝŶĞǀŽůƵƚŝŽŶĂƌǇďŝŽůŽŐǇ͘ ĂƌƌŝŽŶĂŶĚŚŽŽĚĞĚĐƌŽǁƐĂƌĞĂǁĞůůƐƚƵĚŝĞĚĞǆĂŵƉůĞŽĨŝŶĐŝƉŝĞŶƚƐƉĞĐŝĂƚŝŽŶ͕ŝŶǁŚŝĐŚƚǁŽ ƐƵďͲƐƉĞĐŝĞƐ ƌĂƌĞůǇ ŝŶƚĞƌďƌĞĞĚ Žƌ ŽŶůǇ ǁŝƚŚ ůŝƚƚůĞ ƐƵĐĐĞƐƐ͘ ,ŽǁĞǀĞƌ͕ ƚŚĞ ƵŶĚĞƌůǇŝŶŐ ŐĞŶĞƚŝĐ ŵĞĐŚĂŶŝƐŵ ŽĨ ƚŚĞ ŚǇďƌŝĚ ǌŽŶĞ ŝƐ ǇĞƚ ƚŽ ďĞ ƐƚƵĚŝĞĚ ŝŶ ĚĞƚĂŝů͘ ůƚŚŽƵŐŚ ƚŚĞƐĞ ƐƉĞĐŝĞƐ ĂƌĞ ŵŽƌƉŚŽůŽŐŝĐĂůůǇ ĂŶĚ ƚĂǆŽŶŽŵŝĐĂůůǇ ĚŝĨĨĞƌĞŶƚ͕ ƉƌĞǀŝŽƵƐ ƐƚƵĚŝĞƐ ;tŽůĨ Ğƚ Ăů͕͘ ϮϬϭϬͿ ŚĂǀĞ ƐŚŽǁŶƚŚĂƚŵŽůĞĐƵůĂƌĚŝĨĨĞƌĞŶƚŝĂƚŝŽŶďĞƚǁĞĞŶƚŚĞƐƉĞĐŝĞƐŝƐŶŽƚƉƌŽŶŽƵŶĐĞĚĂŶĚŝƐŝŶƐƚĂƌŬ ĐŽŶƚƌĂƐƚƚŽŵŽƌƉŚŽůŽŐŝĐĂůĚŝĨĨĞƌĞŶĐĞƐŝŶƉůƵŵĂŐĞĐŽůŽƌĂƚŝŽŶ͘,ĞŶĐĞ͕ĂŐĞŶŽŵĞǁŝĚĞƐƚƵĚǇ ĐŽƵůĚ ƐĞƌǀĞ ĂƐ Ă ƐƚĂƌƚŝŶŐ ƉŽŝŶƚ ĨŽƌ ŽďƚĂŝŶŝŶŐ Ă ďĞƚƚĞƌ ƵŶĚĞƌƐƚĂŶĚŝŶŐ ŽĨ ƚŚĞ ƉƵƚĂƚŝǀĞůǇ ĨĞǁ ŐĞŶĞƐ ƌĞƐƉŽŶƐŝďůĞ ĨŽƌ ƚŚĞ ŵŽƌƉŚŽůŽŐŝĐĂů ĚŝĨĨĞƌĞŶĐĞƐ͘ tĞ ƉůĂŶ ƚŽ ƵƐĞ ƚŚĞ ĐƌŽǁ ŐĞŶŽŵĞ ĂƐƐĞŵďůǇ ĂƐ Ă ďĂĐŬďŽŶĞ ĨŽƌ ƌĞͲƐĞƋƵĞŶĐŝŶŐ ŽĨ ƐĞǀĞƌĂů ƉŽƉƵůĂƚŝŽŶƐ ŝŶ Ă ƉŽƉƵůĂƚŝŽŶ ŐĞŶŽŵŝĐ ĨƌĂŵĞǁŽƌŬ͘ tĞ ĐŽŵƉĂƌĞĚ ƚŚĞ ƉĞƌĨŽƌŵĂŶĐĞ ŽĨ ƚŚĞ t'^ ĂƐƐĞŵďůǇ ƐƚƌĂƚĞŐǇ ǁŝƚŚ ƚŚĞ ZZ> ĂƐƐĞŵďůǇ ƐƚƌĂƚĞŐǇ ĨŽƌ ƐĞƋƵĞŶĐŝŶŐ ƚŚĞ ĐƌŽǁ ŐĞŶŽŵĞ ƵƐŝŶŐ ŝŶ ƐŝůŝĐŽ ŐĞŶŽŵĞ ƐĞƋƵĞŶĐŝŶŐ ďĂƐĞĚ ŽŶ. 20.

(133) ƐŝŵƵůĂƚŝŽŶƐ͘ tĞ ĂůƐŽ ůŽŽŬĞĚ Ăƚ ĞĨĨĞĐƚŝǀĞŶĞƐƐ ĂŶĚ ĞĂƐĞ ŽĨ ĐŽŶƐƚƌƵĐƚŝŶŐ Ă ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌǇƵƐŝŶŐƌĞƐƚƌŝĐƚŝŽŶĞŶǌǇŵĞƐŝŶƚŚĞůĂďŽƌĂƚŽƌǇ͘ 21.

(134) 3 Materials and methods 3.1 In silico genome sequencing hƐŝŶŐŝŶƐŝůŝĐŽƐŝŵƵůĂƚŝŽŶƐǁĞĐŽŵƉĂƌĞĚƚŚĞt'^ĂŶĚZZ>ŵĞƚŚŽĚŽĨŐĞŶŽŵĞĂƐƐĞŵďůǇ͘dŚĞ t'^ŵĞƚŚŽĚ;&ŝŐƵƌĞϯͿĂƐƐĞŵďůĞƐƐŚŽƌƚƌĞĂĚƐŽďƚĂŝŶĞĚƌĂŶĚŽŵůǇĨƌŽŵƚŚĞĞŶƚŝƌĞŐĞŶŽŵĞ͕ ǁŚŝůĞƚŚĞZZ>ŵĞƚŚŽĚ;&ŝŐƵƌĞϮͿĨŝƌƐƚƉĂƌƚŝƚŝŽŶƐƚŚĞŐĞŶŽŵĞŝŶƚŽƐŵĂůůĞƌĨƌĂĐƚŝŽŶƐǁŚŝĐŚĂƌĞ ƚƌĞĂƚĞĚ ĂƐ ΖŵŝŶŝͲŐĞŶŽŵĞƐΖ͘ :ƵƐƚ ĂƐ ŝŶ ƚŚĞ t'^ ŵĞƚŚŽĚ͕ ƐŚŽƌƚ ƌĞĂĚƐ ĂƌĞ ƌĂŶĚŽŵůǇ ĚƌĂǁŶ ĨƌŽŵ ƚŚĞƐĞ ΖŵŝŶŝͲŐĞŶŽŵĞƐΖ ĂŶĚ ĂƐƐĞŵďůĞĚ ŝŶƚŽ ĐŽŶƚŝŐƐ͘ /Ŷ Ă ƐĞĐŽŶĚ ŵĞƚĂͲĂƐƐĞŵďůǇ ƐƚĞƉ ͚ŵŝŶŝͲŐĞŶŽŵĞƐΖĂƌĞĂƐƐĞŵďůĞĚŝŶƚŽĂĐŽŵƉůĞƚĞŐĞŶŽŵĞ͘ &Žƌ ĐŽŵƉĂƌŝŶŐ ƚŚĞ t'^ ĂŶĚ ZZ> ŵĞƚŚŽĚƐ ǁĞ ŵĂŬĞ ƵƐĞ ŽĨ ƚŚĞ ǌĞďƌĂ ĨŝŶĐŚ ŐĞŶŽŵĞ ĨŽƌ ƉĞƌĨŽƌŵŝŶŐŝŶƐŝůŝĐŽƐŝŵƵůĂƚŝŽŶƐƚŽƐŝŵƵůĂƚĞƚŚĞŬŝŶĚŽĨƌĞƐƵůƚƐƚŚĂƚǁŽƵůĚďĞŽďƚĂŝŶĞĚǁŚŝůĞ ƵƐŝŶŐƚŚĞĐƌŽǁŐĞŶŽŵĞ͘dŚĞǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞŝƐƚŚĞĐůŽƐĞƐƚĂǀĂŝůĂďůĞŐĞŶŽŵĞƐĞƋƵĞŶĐĞ͘ dŚĞ ůĂƚĞƐƚ ǀĞƌƐŝŽŶ ŽĨ ƚŚĞ ǌĞďƌĂ ĨŝŶĐŚ ĚƌĂĨƚ ŐĞŶŽŵĞ ĂƐƐĞŵďůǇ ;th'^ ϯ͘Ϯ͘ϰͬƚĂĞ'ƵƚϭͿ ǁĂƐ ŽďƚĂŝŶĞĚĨƌŽŵƚŚĞh^ǁĞďƐŝƚĞ;ŚƚƚƉ͗ͬͬŐĞŶŽŵĞ͘ƵĐƐĐ͘ĞĚƵͬͿĨŽƌƉĞƌĨŽƌŵŝŶŐĂůůƐŝŵƵůĂƚŝŽŶƐ͘ /ŶƚŚĞĐƌŽǁ͕ƐĂŵƉůĞƐĂƌĞŽďƚĂŝŶĞĚĨƌŽŵǁŝůĚƉŽƉƵůĂƚŝŽŶƐĂƐĐƌŽǁƐĂƐŝƚ͛ƐĚŝĨĨŝĐƵůƚƚŽďƌĞĞĚ ƚŚĞŵ ŝŶ ĐĂƉƚŝǀŝƚǇ ĂŶĚ ŚŝŐŚůǇ ŝŶďƌĞĚ ŝŶĚŝǀŝĚƵĂůƐ ĐĂŶŶŽƚ ďĞ ƉƌŽĚƵĐĞĚ ĚƵĞ ƚŽ Ă ƐƵŝƚĞ ŽĨ ƉƌĂĐƚŝĐĂů ƌĞĂƐŽŶƐ͘ ,ĞŶĐĞ͕ ŝŶƚƌĂͲŝŶĚŝǀŝĚƵĂů ƉŽůǇŵŽƌƉŚŝƐŵƐ ŶĞĞĚ ƚŽ ďĞ ŝŶĐŽƌƉŽƌĂƚĞĚ ĚƵƌŝŶŐ ƚŚĞĂƐƐĞŵďůǇƉƌŽĐĞƐƐ͘E'^ƐĞƋƵĞŶĐŝŶŐĂůƐŽŚĂƐĂŚŝŐŚĞƌƌŽƌƌĂƚĞ;ŽŚŵĞƚĂů͕͘ϮϬϬϴͿ͘/ƚŚĂƐ ďĞĞŶ ƐŚŽǁŶ ƚŚĂƚ ŝŶĐƌĞĂƐĞ ŝŶ ƉŽůǇŵŽƌƉŚŝƐŵ ĂŶĚͬŽƌ ƐĞƋƵĞŶĐŝŶŐ ĞƌƌŽƌ ƉƌŽĚƵĐĞƐ ŵŽƌĞ ĨƌĂŐŵĞŶƚĞĚĂƐƐĞŵďůŝĞƐ;ĂŶŝĞů͘ϮϬϬϵͿǁŚĞŶƵƐŝŶŐƚŚĞt'^ŵĞƚŚŽĚŽĨŐĞŶŽŵĞĂƐƐĞŵďůǇ͘. 22.

(135) ĂƐĞĚŽŶƉƌĞǀŝŽƵƐĞƐƚŝŵĂƚĞƐŽĨƐĞƋƵĞŶĐŝŶŐĞƌƌŽƌĂŶĚƉŽůǇŵŽƌƉŚŝƐŵ;tŽŶŐĞƚů͕͘ϮϬϬϰͿĂůů ƌĞĂĚƐǁĞƌĞƐŝŵƵůĂƚĞĚƵƐŝŶŐĚǁŐƐŝŵǀĞƌƐŝŽŶ͗Ϭ͘ϭ͘Ϯ;ƉĂƌƚŽĨEŶĂůǇƐŝƐWĂĐŬĂŐĞͿǁŝƚŚϬ͘ϭй ƐĞƋƵĞŶĐŝŶŐ ĞƌƌŽƌ ;ďĂƐĞ ĞƌƌŽƌ ƌĂƚĞͿ ĂŶĚ Ϭ͘ϭй ƉŽůǇŵŽƌƉŚŝƐŵ ;ƌĂƚĞ ŽĨ ŵƵƚĂƚŝŽŶƐͿ͘ ZĞƋƵŝƌĞĚ ĐŽǀĞƌĂŐĞǁĂƐŽďƚĂŝŶĞĚƵƐŝŶŐƚŚĞǁƌĂƉƉĞƌƐĐƌŝƉƚ͞ƌĞĂĚƐŝŵ͘Ɖů͟;ƌĞĨĞƌƐƵƉƉůĞŵĞŶƚĂƌǇŵĂƚĞƌŝĂů ĨŽƌƐŽƵƌĐĞĐŽĚĞͿ͘ . 3.1.1 Restriction enzyme and fragment size selection ZĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌŝĞƐĂƌĞĐŽŶƐƚƌƵĐƚĞĚƐŽĂƐƚŽƉĂƌƚŝƚŝŽŶƚŚĞŐĞŶŽŵĞŝŶƚŽƐŵĂůůĞƌ ƐŝǌĞĚ ƌĞƉƌŽĚƵĐŝďůĞ ĨƌĂĐƚŝŽŶƐ ƚŚĂƚ ĂƌĞ ĞĂƐŝĞƌ ƚŽ ŚĂŶĚůĞ ŝŶ ƚŚĞ ĂƐƐĞŵďůǇ ƉƌŽĐĞƐƐ͘ &Žƌ ĞĂĐŚ ĞŶǌǇŵĞ͕ ŝƚ ǁŽƵůĚ ďĞ ŽƉƚŝŵĂů ƚŽ ŚĂǀĞ ĞƋƵĂůůǇ ƐŝǌĞĚ ƉĂƌƚŝƚŝŽŶƐ ŽĨ ƚŚĞ ŐĞŶŽŵĞ ƚŽ ĞŶƐƵƌĞ ƵŶŝĨŽƌŵĚŝƐƚƌŝďƵƚŝŽŶŽĨƌĞĂĚƐ͘tŚĞŶƚŚĞŐĞŶŽŵĞŝƐǇĞƚƚŽďĞƐĞƋƵĞŶĐĞĚŝĚĞŶƚŝĨǇŝŶŐĞŶǌǇŵĞƐ ǁŚŝĐŚ ƉĂƌƚŝƚŝŽŶ ƚŚĞ ŐĞŶŽŵĞ ŝŶƚŽ ĞƋƵĂůůǇ ƐŝǌĞĚ ƉĂƌƚŝƚŝŽŶƐ ĐĂŶ ŽŶůǇ ďĞ ĂĐŚŝĞǀĞĚ ďǇ ƵƐŝŶŐ Ă ĐůŽƐĞůǇƌĞůĂƚĞĚŐĞŶŽŵĞ͘tĞĐŚŽŽƐĞƚŽƵƐĞƚŚĞĐŚŝĐŬĞŶĂŶĚǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞƐǁŚŝĐŚĂƌĞ ĐůŽƐĞůǇ ƌĞůĂƚĞĚ ƚŽ ƚŚĞ ĐƌŽǁ ŐĞŶŽŵĞ͕ ƚŽ ĞŶƐƵƌĞ ƚŚĂƚ ƚŚĞ ĨƌĂŐŵĞŶƚĂƚŝŽŶ ƉĂƚƚĞƌŶ ŝƐ ƌŽďƵƐƚ ĚĞƐƉŝƚĞƚŚĞŵƵůƚŝƉůĞŐĂƉƐŝŶƚŚĞĨŝŶĂůĚƌĂĨƚŐĞŶŽŵĞƐ͘ ƵƌŝŶŐ ůŝďƌĂƌǇ ƉƌĞƉĂƌĂƚŝŽŶ ŝŶ ƚŚĞ ůĂďŽƌĂƚŽƌǇ͕ ĨƌĂŐŵĞŶƚƐ фϭŬď ǁŝůů ďĞ ůŽƐƚ͘ DŽƌĞŽǀĞƌ͕ ǁŝƚŚ ǀĞƌǇůĂƌŐĞĨƌĂŐŵĞŶƚƐ;хϮϬŬďͿŝƚǁŽƵůĚŵĂŬĞŝƚĚŝĨĨŝĐƵůƚƚŽƉĂƌƚŝƚŝŽŶƚŚĞŐĞŶŽŵĞŝŶƚŽϰĞƋƵĂůůǇ ƐŝǌĞĚ ůŝďƌĂƌŝĞƐ͘ dŚĞƌĞĨŽƌĞ͕ ĂŶĞŶǌǇŵĞ ŶĞĞĚĞĚƚŽ ďĞ ƐĞůĞĐƚĞĚ ƐƵĐŚ ƚŚĂƚ ůĞƐƐ ƚŚĂŶ ϱй ŽĨƚŚĞ ŐĞŶŽŵĞǁĂƐƉƌĞƐĞŶƚŝŶĨƌĂŐŵĞŶƚƐфϭŬďŽƌхϮϬŬď͘. 23.

(136) Figure 2: Reduced Representation Library (RRL) approach to genome assembly (Approach taken in this thesis project). 24.

(137) . . Figure 3: Whole Genome Shotgun (WGS) approach to genome assembly. ŝĨĨĞƌĞŶƚ ĞŶǌǇŵĞƐ ĐĂŶ ůĞĂĚ ƚŽ ĚŝĨĨĞƌĞŶƚůǇ ƐŝǌĞĚ ĨƌĂŐŵĞŶƚƐ ďĂƐĞĚ ŽŶ ƚŚĞ ĨƌĞƋƵĞŶĐǇ ŽĨ ƚŚĞ ŽĐĐƵƌƌĞŶĐĞŽĨƚŚĞƌĞƐƚƌŝĐƚŝŽŶƌĞĐŽŐŶŝƚŝŽŶƐŝƚĞ͘dŽƐĞůĞĐƚĞŶǌǇŵĞƐƚŚĂƚĐŽƵůĚďĞƐƵŝƚĂďůĞĨŽƌ ƉƌĞƉĂƌĂƚŝŽŶ ŽĨ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ůŝďƌĂƌŝĞƐ͕ Ăůů ƚŚĞ ϳϱϱ ĞŶǌǇŵĞƐ ĂǀĂŝůĂďůĞ ŝŶ Z^ ǀĞƌƐŝŽŶ ϵϬϵ ǁĞƌĞ ƚĞƐƚĞĚ ĂŐĂŝŶƐƚ ƚŚĞ ĐŚŝĐŬĞŶ ĂŶĚ ǌĞďƌĂ ĨŝŶĐŚ ŐĞŶŽŵĞ͘ ŵŽŶŐ ƚŚĞƐĞ ƚŚĞ ĞŶǌǇŵĞƐǁŚŝĐŚǁĞƌĞŶŽƚƐĞŶƐŝƚŝǀĞƚŽŵĞƚŚǇůĂƚŝŽŶŽƌƉ'ƐŝƚĞƐǁĞƌĞƐĞůĞĐƚĞĚďĂƐĞĚŽŶƚŚĞŝƌ ĂǀĂŝůĂďŝůŝƚǇ ĚƵĞ ƚŽ ŽďƚĂŝŶ ƚŚĞ ĞǆƉĞĐƚĞĚ ƐŝǌĞ ĚŝƐƚƌŝďƵƚŝŽŶ ŽĨ ĨƌĂŐŵĞŶƚƐ ĂŶĚ ĂůƐŽ ĂǀŽŝĚ. 25.

(138) ŚĞƚĞƌŽŐĞŶĞŝƚǇ ŝŶ ƚŚĞ E ĐůĞĂǀĂŐĞ ƌĞĂĐƚŝŽŶ͘ &ŽůůŽǁŝŶŐ ĐĂŶĚŝĚĂƚĞ ĞŶǌǇŵĞƐ ;&ŝŐƵƌĞ ϰͿ ǁĞƌĞ. 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% BglII. BsrGI. EcoRI. PciI. PsiI. PstI. Above 17000 16500 to 17000 16000 to 16500 15500 to 16000 15000 to 15500 14500 to 15000 14000 to 14500 13500 to 14000 13000 to 13500 12500 to 13000 12000 to 12500 11500 to 12000 11000 to 11500 10500 to 11000 10000 to 10500 9500 to 10000 9000 to 9500 8500 to 9000. 8000 to 8500 7500 to 8000 7000 to 7500 6500 to 7000 6000 to 6500 5500 to 6000 5000 to 5500 4500 to 5000 4000 to 4500 3500 to 4000 3000 to 3500 2500 to 3000 2000 to 2500 1500 to 2000 1000 to 1500 500 to 1000 0 to 500. Figure 4: Fragment size distribution for some of the short listed enzymes, namely, BglII, BsrGI, EcoRI, PciI, Psil and Pstl ƐĞůĞĐƚĞĚďĂƐĞĚŽŶƚŚĞƐĞĐƌŝƚĞƌŝĂ͘ ŚŝŐŚ ĐŽƌƌĞůĂƚŝŽŶ ďĞƚǁĞĞŶ ĨƌĂŐŵĞŶƚ ƐŝǌĞ ĚŝƐƚƌŝďƵƚŝŽŶƐ ďĞƚǁĞĞŶ ĐŚŝĐŬĞŶ ĂŶĚ ǌĞďƌĂ ĨŝŶĐŚ ƐƵŐŐĞƐƚƐƚŚĂƚĂƐŝŵŝůĂƌůĞŶŐƚŚƉƌŽĨŝůĞĐĂŶďĞĨŽƵŶĚŝŶƚŚĞĐƌŽǁ;dĂďůĞϮͿ͘ Enzyme. Pearson Correlation (r) calculated using R function cor.test. BglII. 0.91. BsrGI. 0.92. EcoRI. 0.78. PciI. 0.79. PsiI. 0.82. PstI. 0.93. Table 2: Correlation (r) between the fragment size distribution between chicken and zebra finch Genomes. 26.

(139) Figure 5.1: Variation in BglII fragment size distribution at Genomic partitions in human, mouse, chicken and zebra finch genome. Fragment sizes were determined after removing all gaps represented by N’s in the genome.. Figure 5.2: Variation in PciI Fragment size distribution at genomic partitions in human, mouse, chicken and zebra finch genome. Fragment sizes were determined after removing all gaps represented by N’s in the genome. WĐŝ/ ĂŶĚ Őů// ǁĞƌĞ ĨŝŶĂůůǇ ƐĞůĞĐƚĞĚ ĨŽƌ ƚŚĞ ĐŽŶƐƚƌƵĐƚŝŽŶ ŽĨ ƚŚĞ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ůŝďƌĂƌŝĞƐĨŽƌƚŚĞĐƌŽǁŐĞŶŽŵĞĨƌŽŵƚŚĞĐĂŶĚŝĚĂƚĞƐĞƚ;&ŝŐƵƌĞϱͿĚƵĞƚŽƚŚĞŚŝŐŚĚĞŐƌĞĞŽĨ. 27.

(140) ĐŽƌƌĞůĂƚŝŽŶŝŶƚŚĞĨƌĂŐŵĞŶƚƐŝǌĞĚŝƐƚƌŝďƵƚŝŽŶďĂƐĞĚŽŶƐŝŵƵůĂƚŝŽŶĚŽŶĞƵƐŝŶŐƚŚĞĐŚŝĐŬĞŶĂŶĚ ǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞ͘ ZĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌŝĞƐŶĞĞĚƚŽďĞĐŽŶƐƚƌƵĐƚĞĚŝŶƐƵĐŚĂǁĂǇƚŚĂƚƚŚĞŐĞŶŽŵĞŝƐ ĂƉƉƌŽǆŝŵĂƚĞůǇ ĞƋƵĂůůǇ ƉĂƌƚŝƚŝŽŶĞĚ ĂŵŽŶŐ ƚŚĞ ĚŝĨĨĞƌĞŶƚ ůŝďƌĂƌŝĞƐ͘ ĞďƌĂ &ŝŶĐŚ ŝƐ ƚŚĞ ŵŽƐƚ ĐůŽƐĞůǇƌĞůĂƚĞĚƐƉĞĐŝĞƐƚŽƚŚĞĐƌŽǁƚŚĂƚŚĂƐďĞĞŶƐĞƋƵĞŶĐĞĚĂŶĚĂƐƐĞŵďůĞĚ͘,ĞŶĐĞ͕ƚŚĞƐŝǌĞ ƌĂŶŐĞǁŚŝĐŚƉĂƌƚŝƚŝŽŶƐƚŚĞǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞŝŶƚŽĨŽƵƌĞƋƵĂůƉĂƌƚŝƚŝŽŶƐŚĂƐƚŽďĞƵƐĞĚƚŽ ƉĂƌƚŝƚŝŽŶƚŚĞZZůŝďƌĂƌŝĞƐ͘,ŽǁĞǀĞƌ͕ŵĂŶǇůĂƌŐĞŐĂƉƐĂƌĞƉƌĞƐĞŶƚŝŶƚŚĞĞďƌĂ&ŝŶĐŚŐĞŶŽŵĞ͘ dŽ ŽďƚĂŝŶ ƌŽďƵƐƚ ǀĂůƵĞƐ ĨŽƌ ƚŚĞ ĨƌĂŐŵĞŶƚ ƐŝǌĞ Ăůů ĂŶĂůǇƐĞƐ ǁĞƌĞ ƌĞƉĞĂƚĞĚ ǁŝƚŚ ĐŚŝĐŬĞŶ͕ ŚƵŵĂŶĂŶĚŵŽƵƐĞŐĞŶŽŵĞƐƚŽŐĞƚĂŶŝĚĞĂŽĨƚŚĞǀĂƌŝĂƚŝŽŶŝŶƚŚĞĨƌĂŐŵĞŶƚƐŝǌĞĚŝƐƚƌŝďƵƚŝŽŶ ďĞƚǁĞĞŶƚŚĞĚŝĨĨĞƌĞŶƚŐĞŶŽŵĞƐ͘ dŚĞ ĨƌĂŐŵĞŶƚ ƐŝǌĞ ĚŝƐƚƌŝďƵƚŝŽŶ ŝŶ ĐůŽƐĞůǇ ƌĞůĂƚĞĚ ŐĞŶŽŵĞƐ ƐƵĐŚ ĂƐ ŵŽƵƐĞ͕ ŚƵŵĂŶ͕ ĐŚŝĐŬĞŶ ĂŶĚǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞƐǁŝƚŚŽƵƚĂŶǇŐĂƉƐǁĂƐƵƐĞĚƚŽĐŚĞĐŬĨŽƌǀĂƌŝĂƚŝŽŶŝŶĨƌĂŐŵĞŶƚƐŝǌĞƐ͘ ĂƐĞĚ ŽŶ ƐŝŵŝůĂƌŝƚǇ ŝŶ ƚŚĞ ĨƌĂŐŵĞŶƚ ƐŝǌĞ ĚŝƐƚƌŝďƵƚŝŽŶ ŝŶ ĚŝĨĨĞƌĞŶƚ ŐĞŶŽŵĞƐ͕ ĨƌĂŐŵĞŶƚ ƐŝǌĞ ĚŝƐƚƌŝďƵƚŝŽŶŝŶĐŚŝĐŬĞŶǁĂƐƵƐĞĚƚŽƐĞůĞĐƚƚŚĞĐƵƚƐŝƚĞƐĨŽƌWĐŝ/ĂŶĚĨƌĂŐŵĞŶƚƐŝǌĞĚŝƐƚƌŝďƵƚŝŽŶ ŝŶǌĞďƌĂ&ŝŶĐŚǁĂƐƵƐĞĚƚŽƐĞůĞĐƚƚŚĞĐƵƚƐŝƚĞƐĨŽƌŐů//͘ ZĞƐƚƌŝĐƚŝŽŶƐŝƚĞƐǁĞƌĞŝĚĞŶƚŝĨŝĞĚŽŶďŽƚŚƚŚĞƉŽƐŝƚŝǀĞĂŶĚŶĞŐĂƚŝǀĞƐƚƌĂŶĚƐŽĨƚŚĞEƚŽŐĞƚ ƚŚĞ ĨƌĂŐŵĞŶƚ ƐŝǌĞ ĚŝƐƚƌŝďƵƚŝŽŶ͘ ZĞƐƚƌŝĐƚŝŽŶ ƌĞĐŽŐŶŝƚŝŽŶ ƐŝƚĞƐ ;ŝŶ WĐŝ/ d'dͿ ǁĞƌĞ ƵƐĞĚ ƚŽ ŝĚĞŶƚŝĨǇƚŚĞƐŝƚĞƐĂŶĚĐƵƚƐŝƚĞƐ;ŝŶWĐŝ/Ζd'ͿǁĞƌĞƵƐĞĚƚŽŽďƚĂŝŶƚŚĞĚŝĨĨĞƌĞŶƚĨƌĂŐŵĞŶƚƐ͘. 28.

(141) ǆĂĐƚƐŝǌĞƌĂŶŐĞĨŽƌĞĂĐŚůŝďƌĂƌǇǁĂƐĨŽƵŶĚĂƚϳϱй͕ϱϬйĂŶĚϮϱйŽĨƚŚĞƚŽƚĂůŐĞŶŽŵĞƐŝǌĞ ĂĨƚĞƌĞǆĐůƵĚŝŶŐĨƌĂŐŵĞŶƚƐƐŚŽƌƚĞƌƚŚĂŶϭ<͘tŚŝůĞĐƵƚƚŝŶŐŽƵƚƚŚĞĚŝĨĨĞƌĞŶƚůŝďƌĂƌŝĞƐĨƌŽŵ ƚŚĞŐĞůŝŶƚŚĞůĂďŽƌĂƚŽƌǇďĂƐĞĚŽŶƚŚĞƉŽƐŝƚŝŽŶŽĨƚŚĞEůĂĚĚĞƌ͕ĨƌĂŐŵĞŶƚƐŝǌĞƐĐĂŶŽŶůǇ ďĞ ŬŶŽǁŶ ĂƉƉƌŽǆŝŵĂƚĞůǇ͘ ,ĞŶĐĞ͕ ǁĞ ĂůƐŽ ĐŚĞĐŬĞĚ ƚŚĞ ƌŽďƵƐƚŶĞƐƐ ŽĨ ƚŚĞ ůŝďƌĂƌǇ ĨƌĂŐŵĞŶƚ ƐŝǌĞ ďŽƵŶĚĂƌŝĞƐ͘ >ŝďƌĂƌǇ ƐŝǌĞƐ ĂŶĚ ĐŚĂŶŐĞƐ ŝŶ ůŝďƌĂƌǇ ĨƌĂŐŵĞŶƚ ƐŝǌĞ ďŽƵŶĚĂƌŝĞƐ ǁŝƚŚ ƐŵĂůů ĐŚĂŶŐĞƐ ŝŶ ƚŚĞ ůŝďƌĂƌǇ ƐŝǌĞƐ ;dĂďůĞ ϯͿ ƉƌŽǀŝĚĞ ƚŚĞ ŝŶĨŽƌŵĂƚŝŽŶ ŶĞĞĚĞĚ ƚŽ ƉĂƌƚŝƚŝŽŶ ƚŚĞ ŐĞŶŽŵĞ͘ Library (Enzyme). Lower Bound. -5% error. Upper Bound. +5% error. Library-1(PciI). 4.17% (<1kb). 2639(20%). 3090. 3541(30%). Library-2(PciI). 3091. 4996 (45%). 5549. 6139 (55%). Library-3(PciI). 5550. 8357 (70%). 9320. 10548(80%). Library-4(PciI). 9321. NA. 48000. NA. Library-1(BglII). 4.09%(<1kb). 2651(20%). 3106. 3570(30%). Library-2(BglII). 3107. 5077(45%). 5654. 6290(55%). Library-3(BglII). 6291. 8755(70%). 9956. 11525(80%). Library-4(BglII). 11526. NA. 48000. NA. Table 3: Size range for the different libraries digested by different enzymes. 29.

(142) 3.1.2 de novo assembly t'^ŵĞƚŚŽĚŽĨŐĞŶŽŵĞĂƐƐĞŵďůǇ͗ &ŽƌƉĞƌĨŽƌŵŝŶŐƚŚĞt'^ŵĞƚŚŽĚŽĨŐĞŶŽŵĞĂƐƐĞŵďůǇϭϬϬďƉƌĞĂĚƐǁŝƚŚϰϬϬďƉŝŶƐĞƌƚƐŝǌĞ ǁĞƌĞ ŐĞŶĞƌĂƚĞĚ ǁŝƚŚ ĐŽǀĞƌĂŐĞ ŽĨ ϮϬ͕ ϯϬ͕ ϰϬ͕ ϱϬ ĂŶĚ ϲϬ y͘ ĂĐŚ ŽĨ ƚŚĞ ĚĂƚĂ ƐĞƚƐ ǁŝƚŚ ĚŝĨĨĞƌĞŶƚĐŽǀĞƌĂŐĞǁĂƐĂƐƐĞŵďůĞĚƐĞƉĂƌĂƚĞůǇƵƐŝŶŐƚŚĞϲϰďŝƚǀĞƌƐŝŽŶŽĨ^KWĚĞŶŽǀŽ;>ŝĞƚ Ăů͕͘ϮϬϭϬͿŐĞŶŽŵĞĂƐƐĞŵďůĞƌ;ǁŝƚŚϯϭ͕ϲϯĂŶĚϭϮϳŬŵĞƌͿƚƌǇŝŶŐŽƵƚĂůůŬŵĞƌƐŝǌĞƐĨƌŽŵϮϭƚŽ ϲϯĂŶĚƐĞůĞĐƚŝŶŐƚŚĞŬŵĞƌƐŝǌĞǁŝƚŚƚŚĞďĞƐƚEϱϬǀĂůƵĞ͘ ZZ>ŵĞƚŚŽĚŽĨŐĞŶŽŵĞĂƐƐĞŵďůǇ͗ &ŽƌƚŚĞƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌǇĂƉƉƌŽĂĐŚ͕ǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞǁĂƐĚŝŐĞƐƚĞĚŝŶͲƐŝůŝĐŽ ǁŝƚŚ WĐŝ/ ĂŶĚ Őů// ƵƐŝŶŐ ƚŚĞ ͞ZĞƐƚƌŝĐƚŝŽŶ ŝŐĞƐƚŝŽŶ͟ ƉĞƌů ŵŽĚƵůĞ ;ƌĞĨĞƌ ƐƵƉƉůĞŵĞŶƚĂƌǇ ŵĂƚĞƌŝĂů ĨŽƌ ƐŽƵƌĐĞ ĐŽĚĞͿ ƚŽ ŽďƚĂŝŶ ƚŚĞ ĚŝĨĨĞƌĞŶƚ ĨƌĂŐŵĞŶƚƐ ƚŚĂƚ ǁŽƵůĚ ďĞ ŽďƚĂŝŶĞĚ ŝĨ ŐĞŶŽŵŝĐEǁĂƐĂĐƚƵĂůůǇĚŝŐĞƐƚĞĚŝŶƚŚĞůĂďŽƌĂƚŽƌǇ͘dŚĞƐĞĨƌĂŐŵĞŶƚƐǁĞƌĞŐƌŽƵƉĞĚŝŶƚŽϰ ůŝďƌĂƌŝĞƐ ĞĂĐŚ ďĂƐĞĚ ŽŶ ƚŚĞ ƐŝǌĞ ƌĂŶŐĞ ƚŚĂƚ ŚĂƐ ďĞĞŶ ƐĞůĞĐƚĞĚ ;dĂďůĞ ϯͿ͘ /Ŷ ĞĂĐŚ ŽĨ ƚŚĞ ϴ ůŝďƌĂƌŝĞƐ ϭϬϬ ďƉ ƌĞĂĚƐ ǁŝƚŚ ϰϬϬ ďƉ ŝŶƐĞƌƚ ǁĞƌĞ ŐĞŶĞƌĂƚĞĚ ǁŝƚŚ ϮϬ͕ Ϯϱ ĂŶĚ ϯϬ y ĐŽǀĞƌĂŐĞ͘ ĂĐŚ ŽĨ ƚŚĞ ĞŝŐŚƚ ůŝďƌĂƌŝĞƐ ǁĂƐ ĂƐƐĞŵďůĞĚ ƐĞƉĂƌĂƚĞůǇ ƵƐŝŶŐ ƚŚĞ ^KW ĚĞŶŽǀŽ ĂƐƐĞŵďůĞƌ ƉƌŽďŝŶŐĂůůŬŵĞƌƐŝǌĞƐĨƌŽŵϮϭƚŽϲϯ͘&ŽƌĞĂĐŚŽĨƚŚĞĚĂƚĂƐĞƚƐƚŚĞŬŵĞƌǁŝƚŚƚŚĞďĞƐƚEϱϬ ǀĂůƵĞǁĂƐƐĞůĞĐƚĞĚ͘ĞƐƚŬŵĞƌĂƐƐĞŵďůǇĐŽŶƚŝŐƐĨƌŽŵĞĂĐŚŽĨƚŚĞϰůŝďƌĂƌŝĞƐĨŽƌĞĂĐŚĞŶǌǇŵĞ ǁĞƌĞƉŽŽůĞĚƚŽŐĞƚĞŶǌǇŵĞĐŽŶƚŝŐƐ͘ . 30.

(143) dŚĞ Ϯ ƐĞƚƐ ŽĨ ĞŶǌǇŵĞ ĐŽŶƚŝŐƐ ǁĞƌĞ ŵĞƌŐĞĚ ďǇ ŵĞƚĂͲĂƐƐĞŵďůǇ ƵƐŝŶŐ DŝŶŝŵƵƐ ĂƐƐĞŵďůĞƌ ;^ŽŵŵĞƌĞƚĂů͕͘ϮϬϬϳͿĨƌŽŵƚŚĞDK^ƉĂĐŬĂŐĞǁŝƚŚ͞ͲZĞĨĐŽƵŶƚ͟ŽƉƚŝŽŶƐƉĞĐŝĨǇŝŶŐƚŚĞƚǁŽ ƐĞƚƐŽĨĞŶǌǇŵĞĐŽŶƚŝŐƐĂƐƚǁŽĚŝƐƚŝŶĐƚĂƐƐĞŵďůŝĞƐ͘ŽƚŚƉŽƐƐŝďůĞŽƌĚĞƌƐŽĨĂƐƐĞŵďůŝĞƐǁĞƌĞ ƚƌŝĞĚ ĂŶĚ ƚŚĞ ŽƌĚĞƌ ǁŝƚŚ Ă ŚŝŐŚĞƌ EϱϬ ǀĂůƵĞ ǁĂƐ ĐŚŽƐĞŶ͘DŝŶŝŵƵƐϮ ŵĂŬĞƐ ƵƐĞ ŽĨEƵĐŵĞƌ ĨƌŽŵDhDŵĞƌƉĂĐŬĂŐĞ͘dŚĞDhDŵĞƌƉĂĐŬĂŐĞǁĂƐĐŽŵƉŝůĞĚǁŝƚŚƚŚĞϲϰďŝƚŽƉƚŝŽŶƚŽďĞ ĂďůĞƚŽĂƐƐĞŵďůĞůĂƌŐĞĚĂƚĂƐĞƚƐ͘ &ŽƌĞĂĐŚŽĨƚŚĞĚĂƚĂƐĞƚƐƚŚĞŬŵĞƌǁŝƚŚƚŚĞďĞƐƚEϱϬǀĂůƵĞǁĂƐƐĞůĞĐƚĞĚ͘sĂƌŝŽƵƐƐƚĂƚŝƐƚŝĐƐ ƐƵĐŚĂƐEϱϬ͕ůŽŶŐĞƐƚĐŽŶƚŝŐ͕ƚŽƚĂůŶƵŵďĞƌŽĨĂƐƐĞŵďůĞĚďĂƐĞƐ͕ƚŽƚĂůŶƵŵďĞƌŽĨĐŽŶƚŝŐƐǁĂƐ ĐĂůĐƵůĂƚĞĚĨŽƌƚŚĞƐĞĂƐƐĞŵďůŝĞƐƵƐŝŶŐƚŚĞƐĐƌŝƉƚ͞EϱϬ͘Ɖů͟;ƌĞĨĞƌƐƵƉƉůĞŵĞŶƚĂƌǇŵĂƚĞƌŝĂůĨŽƌ ƐŽƵƌĐĞ ĐŽĚĞͿ͘ dŚĞ ĂƐƐĞŵďůĞĚ ĐŽŶƚŝŐƐ ǁĞƌĞ ĂůŝŐŶĞĚ ĂŐĂŝŶƐƚ ƚŚĞ ƌĞĨĞƌĞŶĐĞ ŐĞŶŽŵĞ ƵƐŝŶŐ ŶƵĐŵĞƌĨƌŽŵDhDŵĞƌƉĂĐŬĂŐĞ͘dŚĞƉĞƌĐĞŶƚŽĨƚŚĞŐĞŶŽŵĞƚŚĂƚǁĂƐĂƐƐĞŵďůĞĚ;ƌĞĐŽǀĞƌǇͿ ĂŶĚ ƚŚĞ ĐŽƌƌĞĐƚůǇ ĂƐƐĞŵďůĞĚ ƉƌŽƉŽƌƚŝŽŶ ŽĨ ƚŚĞ ĐŽŶƚŝŐƐ ;ĂĐĐƵƌĂĐǇͿ ǁĞƌĞ ĐĂůĐƵůĂƚĞĚ ƵƐŝŶŐ ΖƐŚŽǁͲƚŝůŝŶŐΖĨƌŽŵDhDŵĞƌƉĂĐŬĂŐĞ͘ . 3.2 Laboratory methods. WƌŝŽƌƚŽƐĞƋƵĞŶĐŝŶŐ͕ŐĞŶŽŵŝĐEŶĞĞĚƐƚŽďĞĞǆƚƌĂĐƚĞĚĂŶĚƉƌĞƉĂƌĞĚŝŶƚŽĂĨŽƌŵƐƵŝƚĂďůĞ ĨŽƌ ƐĞƋƵĞŶĐŝŶŐ ĚĞƉĞŶĚŝŶŐ ŽŶ ƚŚĞ ƐĞƋƵĞŶĐŝŶŐ ƚĞĐŚŶŽůŽŐǇ ďĞŝŶŐ ƵƐĞĚ ĂŶĚ ƐĞƋƵĞŶĐŝŶŐ ƐƚƌĂƚĞŐǇďĞŝŶŐĂĚŽƉƚĞĚ͘. 31.

(144) 3.2.1 DNA extraction and quality check EƌĞƋƵŝƌĞĚĨŽƌŐĞŶŽŵŝĐƐĞƋƵĞŶĐŝŶŐǁĂƐĞǆƚƌĂĐƚĞĚĨƌŽŵĨƌŽǌĞŶďůŽŽĚƐĂŵƉůĞƐƉƌĞƐĞƌǀĞĚ ŝŶYƵĞĞŶΖƐ>ǇƐŝƐďƵĨĨĞƌ͘ZĞĨĞƌƚŽƚŚĞ^ƵƉƉůĞŵĞŶƚĂƌǇŵĂƚĞƌŝĂůĨŽƌĚĞƚĂŝůĞĚƉƌŽƚŽĐŽůƵƐĞĚƚŽ ŽďƚĂŝŶŚŝŐŚƋƵĂůŝƚǇEǁŝƚŚŵŝŶŝŵĂůĨƌĂŐŵĞŶƚĂƚŝŽŶ͘WƵƌŝƚǇĂŶĚƋƵĂŶƚŝƚǇŽĨĞǆƚƌĂĐƚĞĚE ǁĂƐ ŵĞĂƐƵƌĞĚ ďǇ EĂŶŽĚƌŽƉ ĂŶĚ YƵďŝƚ ;/ŶǀŝƚƌŽŐĞŶͿ ŵĞĂƐƵƌĞŵĞŶƚƐ͘ &ŽƌƋƵĂůŝƚǇ ĞǀĂůƵĂƚŝŽŶ͕ E ǁĂƐ ƌƵŶ ŽŶ Ă ϭй ŐĞů ďĞƐŝĚĞ Ă ŚŝŐŚ ŵŽůĞĐƵůĂƌ ǁĞŝŐŚƚ ŵĂƌŬĞƌ ;'Z,ZнKZ ůĂĚĚĞƌ͕ &ĞƌŵĞŶƚĂƐͿ;ĞǆĂŵƉůĞŐĞůƉŝĐƚƵƌĞƐĞĞ&ŝŐƵƌĞϳͿ͘ &ŝŐƵƌĞϳ͗ƌŽǁŐĞŶŽŵŝĐEƐĂŵƉůĞƐĨƌĂŐŵĞŶƚĂƚŝŽŶĐŚĞĐŬĂĨƚĞƌĞǆƚƌĂĐƚŝŽŶĨƌŽŵďůŽŽĚ͘. . 32. .

(145) . 3.2.2 Reduced representation library construction ZĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌŝĞƐǁĞƌĞĐŽŶƐƚƌƵĐƚĞĚƵƐŝŶŐƌĞƐƚƌŝĐƚŝŽŶĞŶǌǇŵĞƐƐĞůĞĐƚĞĚƵƐŝŶŐ ƚŚĞǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞĨŽƌĨŝŶĚŝŶŐĨƌĂŐŵĞŶƚƐŝǌĞĚŝƐƚƌŝďƵƚŝŽŶƐ͘'ĞŶŽŵŝĐEĞǆƚƌĂĐƚĞĚĨƌŽŵ ĐƌŽǁ ďůŽŽĚ ǁĂƐ ĐŚĞĐŬĞĚ ĨŽƌ ƉƵƌŝƚǇ ĂŶĚ ƋƵĂůŝƚǇ ĂƐ ĚĞƐĐƌŝďĞĚ ĂďŽǀĞ͘ ZĞĨĞƌ ƚŽ ƚŚĞ ^ƵƉƉůĞŵĞŶƚĂƌǇ ŵĂƚĞƌŝĂů ĨŽƌ ƚŚĞ ĚĞƚĂŝůĞĚ ƉƌŽƚŽĐŽů ƵƐĞĚ ƚŽ ĐŽŶƐƚƌƵĐƚ ƚŚĞ ƌĞĚƵĐĞĚ ƌĞƉƌĞƐĞŶƚĂƚŝŽŶ ůŝďƌĂƌŝĞƐ͘ WƵƌŝĨŝĞĚ ŐĞŶŽŵŝĐ E ǁĂƐ ĨƵůůǇ ĚŝŐĞƐƚĞĚ ǁŝƚŚ ƚŚĞ ƉƌĞͲƐĞůĞĐƚĞĚ ĞŶǌǇŵĞ;Ğǆ͗Őů//Ϳ͘dŚĞĨƵůůĚŝŐĞƐƚǁĂƐƚŚĞŶƌƵŶŽŶϭйĂŐĂƌŽƐĞŐĞůƚŽƐĞƉĂƌĂƚĞƚŚĞĨƌĂŐŵĞŶƚƐ ďĂƐĞĚŽŶƐŝǌĞ͘ &ŝŐƵƌĞϴ͗ƌŽǁ'ĞŶŽŵŝĐEǁĂƐĚŝŐĞƐƚĞĚǁŝƚŚƚŚĞƐĞůĞĐƚĞĚƌĞƐƚƌŝĐƚŝŽŶĞŶǌǇŵĞĂŶĚƌƵŶŽŶ ĂϭйĂŐĂƌŽƐĞŐĞůĨŽƌƉĂƌƚŝƚŝŽŶŝŶŐƚŚĞŐĞŶŽŵĞŝŶƚŽůŝďƌĂƌŝĞƐďĂƐĞĚŽŶƐŝǌĞ. . . 33.

(146) &ƌŽŵ ƚŚĞ ŐĞŶŽŵŝĐ E ĚŝŐĞƐƚ ĨŽƵƌ ĚŝĨĨĞƌĞŶƚůǇ ƐŝǌĞĚ ;dĂďůĞ ϯͿ ůŝďƌĂƌŝĞƐ ǁĞƌĞ ĐƵƚ ŽƵƚ ĂŶĚ ƉƵƌŝĨŝĞĚ ĨƌŽŵ ƚŚĞ ŐĞů ;ƌĞĨĞƌ ƐƵƉƉůĞŵĞŶƚĂƌǇ ŝŶĨŽƌŵĂƚŝŽŶ ĨŽƌ ĚĞƚĂŝůĞĚ ƉƌŽƚŽĐŽůͿ͘ ŝĨĨĞƌĞŶƚ ŵĞƚŚŽĚƐ;YŝĂŐĞŶ͕'ĞůĂƐĞ͕&ƌĞĞǌĞĂŶĚƐƋƵĞĞǌĞͿĨŽƌĞǆƚƌĂĐƚŝŶŐEĨƌŽŵƚŚĞŐĞůǁĞƌĞƚƌŝĞĚƚŽ ƐĞůĞĐƚĂŵĞƚŚŽĚƚŚĂƚƉƌŽǀŝĚĞĚƚŚĞďĞƐƚǇŝĞůĚǁŝƚŚŵŝŶŝŵĂůĨƌĂŐŵĞŶƚĂƚŝŽŶ;ĚĂƚĂŶŽƚƐŚŽǁŶͿ͘ DƵůƚŝƉůĞŐĞůƐǁĞƌĞƌƵŶƚŽŽďƚĂŝŶŵŽƌĞƚŚĂŶϭϬƵŐŽĨEƉĞƌůŝďƌĂƌǇ͘dŚĞĨŽƵƌůŝďƌĂƌŝĞƐǁĞƌĞ ĂŐĂŝŶƌƵŶŽŶϭйĂŐĂƌŽƐĞŐĞůƚŽĞŶƐƵƌĞƉƌŽƉĞƌƐĞƉĂƌĂƚŝŽŶŽĨƚŚĞĨƌĂŐŵĞŶƚƐŝŶƚŽƚŚĞůŝďƌĂƌŝĞƐ ŽĨƚŚĞƌĞƋƵŝƌĞĚƐŝǌĞ͘ . 34.

(147) 4 Results 4.1 Simulated assemblies. tĞĂƐƐĞŵďůĞĚϭϬϬďƉƌĞĂĚƐǁŝƚŚϰϬϬďƉŝŶƐĞƌƚƐŝǌĞƐŝŵƵůĂƚĞĚĨƌŽŵƚŚĞǌĞďƌĂĨŝŶĐŚŐĞŶŽŵĞ ĨŽƌƚŚĞĞŶƚŝƌĞŐĞŶŽŵĞĂŶĚĨƌŽŵƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌŝĞƐƐĞƉĂƌĂƚĞůǇǁŝƚŚƚŚĞŐŽĂůƚŽ ĐŽŵƉĂƌĞƚŚĞƚǁŽĂƉƉƌŽĂĐŚĞƐŽƵƚůŝŶĞĚĂďŽǀĞ͘ . 4.1.1 Comparison of WGS vs RRL strategy dŚĞƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌǇĂƉƉƌŽĂĐŚŐĂǀĞ;dĂďůĞϰͿůŽŶŐĞƌĐŽŶƚŝŶƵŽƵƐĐŽŶƚŝŐƐ;EϱϬ ƚŚĂƚŝƐϭŬďŵŽƌĞƚŚĂŶƚŚĞt'^ƐĞƋƵĞŶĐŝŶŐƐƚƌĂƚĞŐǇͿ͘,ŽǁĞǀĞƌ͕ƚŚĞƌĞĐŽǀĞƌǇĨƌŽŵƚŚĞt'^ ŵĞƚŚŽĚ ǁĂƐ ϯй ŚŝŐŚĞƌ ǁŚŝůĞ ƚŚĞ ĂĐĐƵƌĂĐǇ ƌĞŵĂŝŶƐ ƚŚĞ ƐĂŵĞ͘ ^ŝŶĐĞ͕ Ă ĚĂƚĂƐĞƚ ǁŝƚŚ ϭй ƐĞƋƵĞŶĐŝŶŐĞƌƌŽƌĂŶĚϭйƉŽůǇŵŽƌƉŚŝƐŵǁĂƐƵƐĞĚĂŶĂĐĐƵƌĂĐǇŽĨϵϵйŝƐĞǆƉĞĐƚĞĚ͘ 4.1.2 Evaluation of RRL library preparation in the laboratory. The construction of the RRL libraries requires the separation of the digested DNA fragments based on size using gel electrophoresis into separate libraries.. Resolution of the high. molecular weight DNA requires the use of low (1 to 0.5%) concentration gels which can be difficult to handle, especially for cutting out bands of various sizes. . 35.

(148) Library. Best Kmer Number of Contigs. Longest Contig. Total number of bases. N50. Recovery. Accuracy. Merged with Minimus (20X +20X). BP (BglII PciI). 81370. 101091. 193094868. 8177. 92.88. 99.9. Merged with Minimus (25X +25X). BP (BglII PciI). 78843. 87214. 192987837. 8443. 92.9. 99.9. Wgs (20X). 47. 3,13,964. 1,10,286. 203077063. 6767. 94.87. 99.93. Wgs (25X). 49. 3,00,744. 1,07,314. 203467969. 6964. 95.19. 99.93. Wgs (30X). 49. 3,02,712. 1,15,974. 203646071. 7022. 95.21. 99.93. Wgs (35X). 49. 3,03,677. 1,03,197. 203751991. 7054. 95.23. 99.93. Wgs (40X). 49. 3,05,229. 1,06,435. 203864219. 7090. 95.24. 99.93. Wgs (45X). 49. 3,06,161. 1,05595. 203940103. 7099. 95.28. 99.93. Wgs (50X). 49. 3,07,288. 1,05,596. 204020912. 7085. 95.27. 99.93. dĂďůĞϰ͗ŽŵƉĂƌŝƐŽŶŽĨtŚŽůĞ'ĞŶŽŵĞ^ĞƋƵĞŶĐŝŶŐǁŝƚŚƚŚĞƌĞĚƵĐĞĚƌĞƉƌĞƐĞŶƚĂƚŝŽŶůŝďƌĂƌǇƐƚƌĂƚĞŐǇďĂƐĞĚŽŶ ƐŝŵƵůĂƚĞĚƉĂŝƌĞĚĞŶĚƌĞĂĚƐ. . Correct size fractions can be cut out from the gel using high molecular weight markers as a reference. However, differences in the concentration of the DNA in the markers and the sample can lead to the marker and sample DNA running at different speeds. It could be seen from repeated tests that the DNA marker was moving master than the digested DNA sample due to its lower concentration. The use of high concentrations of DNA marker leads to smearing of the marker and results in loss of resolution. Ethidium bromide was used due to its compatibility with the downstream sequencing steps. The bands of the appropriate size were cut out from the gel after visualizing them with Ultraviolet light for a short duration. Exposure of DNA to UV leads to degradation and fragmentation of DNA. Using a glass plate to shield from the UV will reduce the damage to. 36.

(149) the DNA. After cutting out the bands from the gel, DNA has to be purified from the gel. Purification of DNA from gel results in fragmentation and loss of DNA. Hence, the method of purification which provides the best yield with least fragmentation needs to be used for purification of DNA from the gel.. Use of Qiagen QIAEX II beads, Gelase and freeze and squeeze methods for purification of DNA from the agarose gel were tried. Qiagen provides the best yields (data not shown) for the low molecular weight libraries while the Gelase method is better for the high molecular weight libraries. However, the ammonium acetate precipitation step in the Gelase method could lead to inactivation of the ligase enzyme while adding the adapters prior to sequencing. All the methods show similar amount of fragmentation of the DNA after purification and provide yields of approximately 50%. Loosing half the DNA in a single purification step requires the use of large amounts of starting material for the RRL approach.. The first step of separation of DNA based on size needs to be verified and validated by at least one more step of size separation. Each step of size separation using gel electrophoresis leads to further fragmentation and loss of DNA. Hence, to obtain a yield of 2 ug DNA for each library, a total of 80 to 100 ug of DNA has to be digested. Digestion and phenol chloroform purification to remove the restriction enzyme leads to a loss of about 20 ug of DNA. The first step of size separation leaves approximately 20 ug of DNA (5 ug/ library). Second step of size separation will give a yield of approximately 2 ug/ library for sequencing. Hence, the RRL approach requires 50 times more DNA than the WGS approach to sequencing.. 37.

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