high-throughput sequencing

Examples of high-throughput sequencing in the following topics:

• Metagenomics

  • 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.
  • To achieve the high coverage needed to fully resolve the genomes of under-represented community members, large samples are needed.
  • The first metagenomic studies conducted using high-throughput sequencing used massively parallel 454 pyrosequencing.
  • These techniques for sequencing DNA generate shorter fragments than Sanger sequencing; 454 pyrosequencing typically produces ~400 bp reads, Illumina and SOLiD produce 25-75 bp reads.
  • An additional advantage to short read sequencing is that this technique does not require cloning the DNA before sequencing, removing one of the main biases in environmental sampling.

• 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

• 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.
  • The later development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
  • 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).

• Multiplex and Real-Time PCR

  • Polymerase Chain Reaction (PCR) is a molecular technique commonly used to amplify nucleic acid sequences.
  • The use of RT-PCR allows for both detection and quantitation of DNA sequences.
  • The key to successful multiplex PCR is the ability to define a single set of reaction parameters (reagent concentrations and cycling parameters) that allows for all primers to anneal with high specificity to their target sequences and be extended with the same efficiency.
  • Multiplex assays can be tedious and time-consuming to establish, requiring lengthy optimization procedures but once optimized numerous high-throughput genomic assays can be achieved at optimum speed.
  • Automated apparatus to amplify DNA sequences using the polymerase chain reaction.

• Microarrays and the Transciptome

  • The study of transcriptomics, also referred to as expression profiling, examines the expression level of mRNAs in a given cell population, often using high-throughput techniques based on DNA microarray technology.
  • Sequencing is now being used instead of gene arrays to quantify DNA levels, at least semi-quantitatively.
  • The core principle behind microarrays is hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs.

• Total Quality Management

  • In contrast, cell layouts promote JIT goals by featuring unidirectional product flows, high visibility, and fast throughput times.

• Introduction to negative binomial distribution

  • Each day a high school football coach tells his star kicker, Brian, that he can go home after he successfully kicks four 35 yard field goals.
  • OR 10th sequence)
  • P(1st sequence OR 2nd sequence OR ...
  • OR 10th sequence) = P(1st sequence) + P(2nd sequence) +·+ P(10th sequence)
  • P(it takes Brian six tries to make four field goals) = [Number of possible sequences]×P(Single sequence)

• 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.

• Autoimmune Diseases

  • Modern high throughput technologies, like mRNA microarrays, have enabled researchers to investigate diseases at a genome-wide level.

• Uniform plant loading, flexible resources, and line/cellular flow layouts

  • In contrast, cell layouts promote JIT goals by featuring unidirectional product flows, high visibility, and fast throughput times.