Shotgun sequencing

shotgunwhole genome shotgun sequencingpaired end sequencingshotgun methodwhole-genome shotgunbottom-up sequencingcommunity sequencingcoveragecurrent DNA sequencing technologygenome sequencing
In genetics, shotgun sequencing is a method used for sequencing random DNA strands.wikipedia
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Sequence assembly

genome assemblyassembledassembly
Due to this size limit, longer sequences are subdivided into smaller fragments that can be sequenced separately, and these sequences are assembled to give the overall sequence.
Typically the short fragments, called reads, result from shotgun sequencing genomic DNA, or gene transcript (ESTs).

Whole genome sequencing

genome sequencingfull genome sequencingsequenced
Shotgun sequencing was one of the precursor technologies that was responsible for enabling full genome sequencing.
influenzae'', were sequenced by Shotgun sequencing.

DNA sequencing

DNA sequencesequencesequencing
The chain termination method of DNA sequencing ("Sanger sequencing") can only be used for short DNA strands of 100 to 1000 base pairs.
The different strategies have different tradeoffs in speed and accuracy; shotgun methods are often used for sequencing large genomes, but its assembly is complex and difficult, particularly with sequence repeats often causing gaps in genome assembly.

Human Genome Project

human genomeELSIsequencing of the human genome
For example, to complete the Human Genome Project, most of the human genome was sequenced at 12X or greater coverage; that is, each base in the final sequence was present on average in 12 different reads.
Each of these pieces was then sequenced separately as a small "shotgun" project and then assembled.

Primer walking

chromosome walkinggenome walkingChromosome-walking
Primer walking (or "chromosome walking") progresses through the entire strand piece by piece, whereas shotgun sequencing is a faster but more complex process that uses random fragments.
Primer walking was also the basis for the development of shotgun sequencing, which uses random primers instead of specifically chosen ones.

DNA sequencing theory

DNA sequencingLander-Waterman formulapaired-end sequencing
Broader application benefited from pairwise end sequencing, known colloquially as double-barrel shotgun sequencing.
This whole-genome sequencing method became immensely popular as it was championed by Celera and used to sequence several model organisms before Celera applied it to the human genome.

Contig

contigscontig mapBAC contigs
Because multiple genome copies have been sheared at random, the fragments contained in these clones have different ends, and with enough coverage (see section above) finding a scaffold of BAC contigs that covers the entire genome is theoretically possible.
In bottom-up sequencing projects, a contig refers to overlapping sequence data (reads); in top-down sequencing projects, contig refers to the overlapping clones that form a physical map of the genome that is used to guide sequencing and assembly.

Bacterial artificial chromosome

BACBACsbacterial artificial chromosomes
If the gap is large (>20kb) then the large fragment is cloned in special vectors such as bacterial artificial chromosomes (BAC) followed by sequencing of the vector.
BACs were replaced with faster and less laborious sequencing methods like whole genome shotgun sequencing and now more recently next-gen sequencing.

Haemophilus influenzae

H. influenzaeHaemophilus influenzae type BHemophilus influenzae
The strategy was subsequently adopted by The Institute for Genomic Research (TIGR) to sequence the genome of the bacterium Haemophilus influenzae in 1995, and then by Celera Genomics to sequence the Drosophila melanogaster (fruit fly) genome in 2000, and subsequently the human genome.
The sequencing method used was whole-genome shotgun, which was completed and published in Science in 1995.

Sequence-tagged site

sequence tagged sites (STS)sequence tags
A small radioactively or chemically labeled probe containing a sequence-tagged site (STS) can be hybridized onto a microarray upon which the clones are printed.
They are used in shotgun sequencing, specifically to aid sequence assembly.

Clinical metagenomic sequencing

clinical use
The sensitivity of metagenomic sequencing makes it an attractive choice for clinical use.
The untargeted analysis is a metagenomic "shotgun" approach.

Genetics

geneticgeneticistgenetically
In genetics, shotgun sequencing is a method used for sequencing random DNA strands.

Sequencing

sequencedsequencemolecular data
In genetics, shotgun sequencing is a method used for sequencing random DNA strands.

DNA

deoxyribonucleic aciddouble-stranded DNAdsDNA
In genetics, shotgun sequencing is a method used for sequencing random DNA strands.

Shotgun

shotguns12-gauge12 gauge
It is named by analogy with the rapidly expanding, quasi-random firing pattern of a shotgun.

Base pair

base pairsbpMbp
The chain termination method of DNA sequencing ("Sanger sequencing") can only be used for short DNA strands of 100 to 1000 base pairs.

Repeated sequence (DNA)

repetitive DNArepeat sequencesrepeated sequences
Assembly of complex genomes is additionally complicated by the great abundance of repetitive sequences, meaning similar short reads could come from completely different parts of the sequence.

Euchromatin

euchromatic
Even so, current methods have failed to isolate or assemble reliable sequence for approximately 1% of the (euchromatic) human genome, as of 2004.

Cauliflower mosaic virus

CaMVCaMV35SCauliflower mosaic virus (CaMV)
The first genome sequenced by shotgun sequencing was that of cauliflower mosaic virus, published in 1981.

Hypoxanthine-guanine phosphoribosyltransferase

HPRTHPRT1HGPRT
The first published description of the use of paired ends was in 1990 as part of the sequencing of the human HGPRT locus, although the use of paired ends was limited to closing gaps after the application of a traditional shotgun sequencing approach.

J. Craig Venter Institute

The Institute for Genomic ResearchInstitute for Genomic ResearchTIGR
The strategy was subsequently adopted by The Institute for Genomic Research (TIGR) to sequence the genome of the bacterium Haemophilus influenzae in 1995, and then by Celera Genomics to sequence the Drosophila melanogaster (fruit fly) genome in 2000, and subsequently the human genome.

Celera Corporation

Celera GenomicsCeleraCelera Diagnostics
The strategy was subsequently adopted by The Institute for Genomic Research (TIGR) to sequence the genome of the bacterium Haemophilus influenzae in 1995, and then by Celera Genomics to sequence the Drosophila melanogaster (fruit fly) genome in 2000, and subsequently the human genome.

Drosophila melanogaster

fruit fliesDrosophilafruit fly
The strategy was subsequently adopted by The Institute for Genomic Research (TIGR) to sequence the genome of the bacterium Haemophilus influenzae in 1995, and then by Celera Genomics to sequence the Drosophila melanogaster (fruit fly) genome in 2000, and subsequently the human genome.

Molecular cloning

cloneclonedrecombinant DNA technology
To apply the strategy, a high-molecular-weight DNA strand is sheared into random fragments, size-selected (usually 2, 10, 50, and 150 kb), and cloned into an appropriate vector.

Vector (molecular biology)

vectorvectorsVector DNA
To apply the strategy, a high-molecular-weight DNA strand is sheared into random fragments, size-selected (usually 2, 10, 50, and 150 kb), and cloned into an appropriate vector.