shotgun sequencing

(noun)

A DNA sequencing technique in which a large number of small fragments of a long DNA strand are generated at random, sequenced, and reassembled to form a sequence of the original strand.

Related Terms

Examples of shotgun sequencing in the following topics:

  • Detecting Uncultured Microorganisms

    • These advances have allowed the adaptation of shotgun sequencing to metagenomic samples .
    • The approach, used to sequence many cultured microorganisms and the human genome, randomly shears DNA, sequences many short sequences, and reconstructs them into a consensus sequence.
    • Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples.
    • This was further followed by high-throughput sequencing which did the same process as the shotgun sequencing but at a much bigger scale in terms of the amount of DNA that could sequenced from one sample.
    • (A) sampling from habitat; (B) filtering particles, typically by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E) sequencing the clones; (F) sequence assembly into contigs and scaffolds

  • Metagenomics

    • Recent studies use "shotgun" Sanger sequencing or massively parallel pyrosequencing to get largely unbiased samples of all genes from all the members of the sampled communities.
    • Advances in bioinformatics, refinements of DNA amplification, and the proliferation of computational power have greatly aided the analysis of DNA sequences recovered from environmental samples, This allows the adaptation of shotgun sequencing to metagenomic samples.
    • Shotgun sequencing and screens of clone libraries reveal genes present in environmental samples.
    • Shotgun metagenomics is also capable of sequencing nearly complete microbial genomes directly from the environment.
    • On the other hand, the random nature of shotgun sequencing ensures that many of these organisms, which would otherwise go unnoticed using traditional culturing techniques, will be represented by at least some small sequence segments.

  • Genetic Analysis

    • There are 32 microbial genomes sequenced to date and published (25 domain Bacteria, 5 Domain Archaea, 1 domain Eukarya).
    • There are two main approaches to sequencing microbial genomes – the ordered clone approach and direct shotgun sequencing both require large and small insert genomic DNA libraries in order to be effective.
    • PCR products of every gene from a complete genome sequence are bound in a high-density array on a glass slide.
    • Mutation is random, undirected, heritable variation caused by alteration in nucleotide sequence at some point of DNA.
    • Summarize the techniques used to study genomes: PFGE. ordered clone approach, direct shotgun sequencing and microarray hybridization

  • Viral Genomes in Nature

    • Metagenomic sequencing is particularly useful in the study of viral communities.
    • In 2002, Mya Breitbart, Forest Rohwer, and colleagues used environmental shotgun sequencing to show that 200 liters of seawater contains over 5,000 different viruses.
    • (A) sampling from habitat; (B) filtering particles, typically by size; (C) Lysis and DNA extraction; (D) cloning and library construction; (E) sequencing the clones; (F) sequence assembly into contigs and scaffolds

  • DNA Sequencing Based on Sanger Dideoxynucleotides

    • Sanger sequencing, also known as chain-termination sequencing, refers to a method of DNA sequencing developed by Frederick Sanger in 1977.
    • More recently, dye-terminator sequencing has been developed.
    • Automated DNA-sequencing instruments (DNA sequencers) can sequence up to 384 DNA samples in a single batch (run) in up to 24 runs a day.
    • Automation has lead to the sequencing of entire genomes.
    • Different types of Sanger sequencing, all of which depend on the sequence being stopped by a terminating dideoxynucleotide (black bars).

  • DNA Sequencing of Insertion Sites

    • An insertion sequence (also known as an IS, an insertion sequence element, or an IS element) is a short DNA sequence that acts as a simple transposable element.
    • The coding region in an insertion sequence is usually flanked by inverted repeats.
    • Although insertion sequences are usually discussed in the context of prokaryotic genomes, certain eukaryotic DNA sequences belonging to the family of Tc1/mariner transposable elements may be considered to be insertion sequences.
    • A complex transposon does not rely on flanking insertion sequences for resolvase.
    • It involves cutting genomic DNAs with a restriction enzyme, ligating vectorettes to the ends, and amplifying the flanking sequences of a known sequence using primers derived from the known sequence along with a vectorette primer.

  • Slipped-Strand Mispairing

    • Slipped strand mispairing (SSM) is a process that produces mispairing of short repeat sequences during DNA synthesis.
    • The short repeat sequences are 1 to 7 nucleotides and can be homogeneous or heterogeneous repetitive DNA sequences.
    • The overlapping promoter regions have repeats of the dinucleotide TA in the -10 and -35 sequences.
    • The second way that SSM induces transcriptional regulation is by changing the short repeat sequences located outside the promoter.
    • Black boxes are short sequence repeats.

  • Homologs, Orthologs, and Paralogs

    • The terms "percent homology" and "sequence similarity" are often used interchangeably.
    • As with anatomical structures, high sequence similarity might occur because of convergent evolution, or, as with shorter sequences, because of chance.
    • Such sequences are similar, but not homologous.
    • Sequence regions that are homologous are also called conserved.
    • Paralogous sequences provide useful insight into the way genomes evolve.

  • Mutation

    • Mutations are accidental changes in a genomic sequence of DNA; this includes the DNA sequence of a cell's genome or the DNA or RNA sequence.
    • In molecular biology and genetics, mutations are accidental changes in a genomic sequence of DNA: the DNA sequence of a cell's genome or the DNA or RNA sequence in some viruses.
    • These random sequences can be defined as sudden and spontaneous changes in the cell.
    • In general, this form of mutagenesis requires that the wild type gene sequence be known.

  • Vectors for Genomic Cloning and Sequencing

    • The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
    • Vectors called expression vectors (expression constructs) are specifically for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene.
    • Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell .
    • These plasmid transcription vectors characteristically lack crucial sequences that code for polyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible.
    • However, because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection.