"> Genome Sequencing Approaches – Course Writing Pals

Genome Sequencing Approaches


During the aftermost 100 years, medical assay grew from a simple animal concern for a few attenuate ancestral disorders, to a accustomed medical specialty with concepts and approaches that are key elements for the assay and administration of abounding disorders, both accepted and attenuate (Thompson & Thompson). Numerous DNA techniques in alongside with affiliation and bond studies accept abundantly contributed to the identification of causal abiogenetic factors of animal disorders. Notwithstanding, a lot of questions remained changing (Vogel & Motulsky, 2010b).

Development of technologies that admittance absolute assay of accustomed and aberrant genes and the announcement of genes in both accustomed and ache states added our adeptness to accept bigger the abstraction of atomic assay at all levels, from the gene to the accomplished animal (Thompson & Thompson). Particularly, the contempo developments in the acreage of DNA sequencing technologies accept revolutionized the animal assay and genomics in advantageous and ache (Motulsky, 2010b). No doubt, over the advancing decades, abiogenetic ache will be abundant bigger accepted as a aftereffect of abiogenetic assay (Griffiths).

The Animal Genome

In adjustment to be able to accept the biological acceptation of abiogenetic advice in bloom and disease, we charge aboriginal apperceive the accomplished nucleotide adjustment of the animal genome (Vogel &Motulsky, Antonarakis). Nowadays, we apperceive that the animal genome comprises two audible parts: a circuitous nuclear genome and a actual simple mitochondrial genome.

The advertisement of the mitochondrial DNA adjustment of the animal genome in 1981, led to a abundant compassionate of its accent and function. Mitochondrial DNA (mtDNA) consists of a blazon of annular double-stranded DNA, absolute 37 genes that lie in a absolute breadth of 16.6 kilobases in length. The beef about accommodate bags of copies of the mtDNA atom and their cardinal can adapt appreciably in altered corpuscle types (Strachan).

In adverse with the mtDNA, the adaptation of the added circuitous nuclear genome has been a added arduous appointment (Strachan). Thus an all-embracing collaborative project, alleged ‘’The Animal Genome Project’’(HGP), was accomplished on 1 October 1990, accepting a audible purpose to actuate the nucleotide adjustment of the animal genome. In 2004, the HGP was about completed (Vogel & Motulsky) and admitting the aerial amount of this attempt, about US$ 3billion (www.genome.gov), the abeyant medical allowances from the adeptness of the animal genome adjustment were the aloft motive abaft the allotment of this all-embracing project. The four capital objectives of the HGP were to: actuate the nucleotide adjustment of the genome; assemble a concrete map of the animal genome; assemble the bond map of the animal genome; and accommodate an antecedent assay of the aberration amid animal genomes (Vogel & Motulsky).

The huge absorption about the about adjourned HGP met with antagonistic antagonism from abreast adjourned genome sequencing projects. In 1999, the Celera aggregation appear that it advised to aftermath a abstract adjustment of the animal genome in abbreviate aeon of two years (Strachan) and in actuality this arrest may accept provided the all-important antagonism for the accurate achievement of the HGP (Vogel & Motulsky). The two altered projects accept followed altered cardinal approaches: The HGP Accumulation acclimated hierarchical shotgun sequencing (figure 1A), admitting the Celera accumulation acclimated the whole-genome shotgun sequencing access (figure 1B). In hierarchical shotgun sequencing approach, DNA libraries are mapped into a concrete map on the chromosomes and again alone clones are alleged for sequencing. The accumulation of these sequences leads to the architecture of the animal genome (International Animal Genome Sequencing Consortium, 2004). The whole-genome shotgun sequencing access is additionally based on the architecture of plasmid libraries followed by absolute sequencing of the bits and accumulation in connected sequences (Venter et al., 2001).


Figure 1: Genome sequencing approaches. A: In accomplished genome shotgun sequencing, the absolute genome is sheared about into baby bits (appropriately sized for sequencing) and again reassembled. B: In hierarchical shotgun sequencing, the genome is aboriginal burst into beyond segments.

In 2004 the achievement of “The Animal Genome Project” appear the 99% of the euchromatic adjustment of the animal genome, to the aerial accurateness of 1 absurdity in 100,000 nucleotides (Vogel & Motulsky, Antonarakis, 2010).

Once the antecedent abstract animal genome adjustment had been obtained, all-embracing efforts accept been focused on the identification and assuming of functionally important sequences, such genes and authoritative elements. Through 2004, about 21.000 genes accept been absolutely characterized by free the coding DNA sequences (CDS). Admitting that, there are still ample ambiguities about the exact cardinal of the genes we accept (Strachan).

Today, the animal genome complexity, advance to a absolute breadth of 3,384,269,757 abject pairs (Genome Reference Consortium GRCh38.p2), makes the assurance of the abiogenetic abject for bloom and diseases a massive claiming for scientists.

Genetic Disorders

A ache can be characterized as the alternation amid genes and environment. However, the about role of the abiogenetic agency may be baby or large. Therefore a ataxia is acquired wholly or partly by abiogenetic factors. Traditionally, abiogenetic disorders are classified in three categories: chromosome disorders, single-gene disorders, and multifactorial disorders (Thompson & Thompson).

The accepted estimated cardinal for the bearing prevalence of breed with austere complete disorders is 53 out of 1000 alive births. From these, 3.9 out of 1000 are chromosomal disorders, 16.8 out of 1000 single-gene disorders and the rest, multifactorial disorders or of alien agent (Vogel & Motulsky).

WHO region

Population millions

Chromosomal disorders/1.000

Single gene disorders/1.000

Total complete disorders/1000

Annual afflicted alive births

Eastern Mediterranean












SE Asia


















Western Pacific












Table 1: Overall all-around abstracts for the abundance of abiogenetic disorders, by WHO (Vogel & Motulsky).

Thence, the assuming of mutations advance to abiogenetic disease, the compassionate how these mutations affect bloom and the acceptance of this advice to advance assay and administration of diseases, are the arch aims of avant-garde medical assay (Thompson & Thompson).

Mendelian disorders

A ataxia is characterized as Mendelian back the phenotypic affection of the ache is transmitted by a distinct locus afterward the laws of allegory and absolute array (Vogel & Motulsky, Speicher, 2010). Considering the actuality that bodies are diploid organisms, all the alleles should chase the laws of Mendelian inheritance. Though, the majority of animal abiogenetic characters are not advised as Mendelian. They are absolute by added than one gene locus. The abiogenetic assurance may absorb abounding loci (polygenic), a baby cardinal of loci (oligogenic) or a distinct locus with a polygenic background. Generally, the added circuitous the alleyway amid a DNA adjustment and a phenotypic trait, the beneath acceptable it is that the affection will appearance a Mendelian full-blooded adjustment (Strachan).

Basically, there are bristles patterns of Mendelian inheritance: autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, Y-linked. Since males accept alone a distinct archetype of X and Y chromosomes, they are hemizygous for anniversary gene locus on the X and Y chromosomes. Therefore, they are never heterozygous for any X-linked or Y-Iinked appearance and in adjustment to adumbrate a male’s phenotype from his genotype it is not appropriate to apperceive whether this appearance is backward or ascendant (Strachan).

Analyzing a archetypal of bequest that follows the Mendel’s laws seems to be an accessible assignment. However, in absolute activity a basal Mendelian adjustment can be buried by assorted complications that are analyzed beneath (Strachan). For instance, some disorders it is accessible to not be bidding at all in an individual, admitting this being accepting the aforementioned genotype that causes the ataxia in alternative associates of his family. Moreover, the phenotypic announcement of an aberrant genotype may be adapted by altered furnishings of aging, alternative abiogenetic loci, or accidental ecology effects. (Thompson & Thompson). Added specifically, phenomena such as penetrance (the admeasurement of individuals that appearance at atomic some amount of announcement of the aberrant genotype) (Cummings), expressivity (the severity of announcement of the phenotype amid individuals with the aforementioned ache causing genotype) (Thompson&Thompson), anticipation (the addiction of some altitude to become added astringent or accept beforehand access in alternating generations), imprinting (autosomal ascendant characters that are bidding alone back affiliated from a ancestor of one accurate sex) (Strachan), genetic heterogeneity (different mutations at the aforementioned locus, allelic heterogeneity, or mutations at altered loci, locus heterogeneity, aftermath the aforementioned phenotype), phenotypic heterogeneity (different mutations in the aforementioned gene, accord acceleration to conspicuously altered phenotypes), can generally complicate the assay of diseases and the estimation of ancestors pedigrees (Thompson & Thompson).

Molecular assay and carrier testing in a accommodating and his or her ancestors requires the identification of the adroit alteration for a Mendelian disease, a footfall of abundant accent for accommodating administration and ancestors counseling (Gilissen, 2011). Beyond this, identification of such monogenic disorders contributes abundantly to our compassionate of gene functions and biological pathways associated with bloom and ache in accepted (Oti, 2007). The actuality that abundant of our compassionate about the abiogenetic abject of altered circuitous diseases is based on our adeptness of Mendelian disorders, reflects the accent of belief these monogenic disorders (Vogel & Motulsky, Clark). Finally, abiogenetic phenomena such as uniparental disomy, affectionate imprinting and epistatic interactions accept been declared through studies relied on the assay of Mendelian ancestry (Antonarakis, 2006).

Originally, applicant gene resequencing and acceptable gene mapping approaches (Table 2) such as karyotyping, bond analysis, homozygosity mapping and archetype cardinal variants (CNV) assay aided the comment of the abiogenetic abject of abounding accepted and attenuate monogenic disorders (Gilissen, 2011). Over the antecedent decade, the addition of new technologies that accredit the sequencing of DNA at college throughput amount and at abundant lower cost, comparing to earlier techniques, gave a addition to the assay of monogenic disorders (Shendure, 2008) and accustomed a affluent framework for advertent the genes basal ahead baffling Mendelian disorders (Bamshad, 2011).


Applies to



Candidate gene

Any disease

Easy to accomplish for one or two genes; requires no mapping, can anon analyze the adroit variant/mutation

Relies heavily on accepted biological knowledge; success amount actual low

Genetic mapping by karyotyping

Any disease

Easy to perform; no familial cases required; can ascertain (large) counterbalanced events

Low resolution, alone detects ample chromosomal aberrations; alteration apprehension requires additional step

Genetic mapping by bond analysis

Inherited disease

Easy to perform

Requires ample families, generally identifies ample loci; alteration apprehension requires additional step

Genetic mapping by homozygosity mapping

Recessive monogenic diseases

Small families can be used

Most advantageous for affiliated families; generally identifies ample loci; alteration apprehension requires additional step

Genetic mapping by CNV analysis

Monogenic/monolocus disease

High resolution CNV screening; no familial cases required; can potentially analyze baby loci

Only investigates CNVs; cannot ascertain counterbalanced events, no base-pair resolution; alteration apprehension requires additional step

Whole exome sequencing (WES)

Any disease

Base-pair resolution exome-wide; detects best types of genomic variation; can anon analyze the adroit variant/mutation

Unable to ascertain non-coding variants; bound resolution for CNVs and alternative structural variation; advantage airheadedness due to accessory process; almost expensive

Whole genome sequencing (WGS)

Any disease

Base-pair resolution genome-wide; detects all types of genomic variation; can anon analyze the adroit variant/mutation

Data assay complex; alike added big-ticket than exome sequencing

Table 2: Mendelian ache gene identification approaches (Gilissen, 2011)

Human abiogenetic aberration and mutations

Human genome variation

The adjustment of nuclear DNA is about 99.9% identical amid any two humans. It is alone a baby atom of DNA adjustment that differs amid individuals, which is amenable for the abiogenetic airheadedness amid animal citizenry (Thompson & Thompson). This abiogenetic airheadedness is the atomic substrate of the evolutionary action (Vogel&Motulsky). Distinct nucleotide variants (SNVs), segmental duplications, low-copy repeats, indels (insertions, deletions), inversions and archetype cardinal variants (CNVs) can actuate the abiogenetic aberration of the animal genome (Vogel & Motulsky, Antonarakis, 2010). Back a alternative is so accepted that it is begin in added than 1% of chromosomes in the accepted population, again it is characterized as a abiogenetic polymorphism. In contrast, attenuate variants accept frequencies beneath than 1% in the accepted population. Abounding deleterious mutations that advance to abiogenetic ache are attenuate variants. However, there is not a simplified aphorism to call the accord amid the aftereffect of the allele on bloom and its frequency. Lots of attenuate variants appear to accept no deleterious effect, admitting some variants accepted abundant to be polymorphisms are accepted to activate to austere affliction (Thompson&Thompson). For example, the commonest alteration that causes cystic fibrosis in arctic European populations (p.F508del) has a abundance of 1-2 % in arctic European populations (Strachan).

A arch footfall in animal citizenry assay over the accomplished decade was the HapMap project, an all-embracing accomplishment committed to the conception of a genome-wide haplotype map. Hundreds of bags of SNPs and microsatellites accept been articular by genotyping accidental populations all over the world. The aftereffect is a awful abundant addle of aberration in our species. The abstracts are accessible to the accessible at several Web sites, including that of the All-embracing HapMap Activity (www.hapmap.org) and the Animal Genome Diversity Activity (hgdp.uchicago.edu) (Griffiths).


Mutations are the ultimate antecedent of all abiogenetic aberration (Griffiths). Any change in the nucleotide adjustment or adjustment of DNA is authentic as mutation. Traditionally, they are classified into three capital categories (Table 3): genome mutations (affect the cardinal of chromosomes in the cell), chromosome mutations (alter the anatomy of alone chromosomes) and gene mutations (alter alone genes).

Class of Mutation


Frequency (Approximate)


Genome mutation

Chromosome missegregation

2-4 × 102/cell division


Chromosome mutation

Chromosome rearrangement

6 × 104/cell division


Gene mutation

Base brace mutation,

small indels

10-4/base pair/cell division

Point mutations

Table 3: Types of Alteration and Their Estimated Frequencies (Thompson & Thompson)

Genome mutations are alterations in the cardinal of complete chromosomes, alleged aneuploidy. They appear from errors in chromosome allegory during meiosis or mitosis. Chromosome mutations are changes that accuse alone a allotment of a chromosome, such as fractional duplications or triplications, deletions, inversions, and translocations. They action spontaneously or may aftereffect from aberrant allegory of translocated chromosomes during meiosis. Gene mutations are declared as changes in the DNA adjustment of the mitochondrial or nuclear genomes, alignment from a change in a distinct nucleotide to changes that affect millions of abject pairs. They can action by errors during the action of DNA replication, or from a abortion to adjustment DNA afterwards damage. All three types of alteration action at ample frequencies in abounding altered cells. In case that a alteration crops up in germline corpuscle populations, it is accessible be anesthetized on to abutting generations. Contrariwise, actual mutations action accidentally alone in a subset of beef in assertive tissues, consistent in actual mosaicism (i.e. cancer), appropriately they can not be transmitted to the abutting generation. Mutations can appear spontaneously or induced by actinic agents alleged mutagens, due to their adeptness to enhance the amount of mutations (Thompson & Thompson).

Gene mutations and pathogenic variants

As mentioned above, mutations, and abnormally gene mutations, are amenable for the abiogenetic aberration amid animal populations and represent the ultimate antecedent of evolutionary changes (Olson-Manning, 2012). The all-inclusive majority of these mutations are absolutely controllable and accept no aftereffect on the genome fitness; but alike those that do affect the phenotype are advised allotment of the accustomed aberration that makes us all individual. Thence, medical apropos focus to those variants that are authentic as pathogenic and associated with altered disorders. Nonetheless, some of these variants may be pathogenic beneath specific altitude such as the ecology stress, and others may accept bashful furnishings that apparent as susceptibility to a ache alone in aggregate with alternative abiogenetic variants (Strachan).

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