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.

• 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.

• 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.
  • The level of cDNA is then quantified using high-resolution image scanners.
  • Mutation is random, undirected, heritable variation caused by alteration in nucleotide sequence at some point of DNA.

• Regulation by Biosynthetic Enzymes

  • Attenuator is a nucleotide sequence in DNA that can lead to premature termination of transcription.
  • When there is a high level of tryptophan in the region, it is inefficient for the bacterium to synthesize more.
  • (This differs from eukaryotic cells, where RNA must exit the nucleus before translation starts. ) The attenuator sequence, which is located between the mRNA leader sequence (5' UTR) and trp operon gene sequence, contains four domains, where domain 3 can pair with domain 2 or domain 4.
  • A high level of tryptophan will permit ribosomes to translate the attenuator sequence domains 1 and 2, allowing domains 3 and 4 to form a hairpin structure, which results in termination of transcription of the trp operon.
  • The attenuator sequence has its codons translated into a leader peptide, but is not part of the trp operon gene sequence.

• Synthesizing DNA

  • DNA must be synthesized to study genes, the sequence of genomes, and many other studies.
  • Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids, both DNA and RNA with a defined chemical structure (sequence).
  • The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence.
  • To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product.
  • Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity.

• Size Variation and ORF Contents in Genomes

  • Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop codons.
  • Long ORFs are often used, along with other evidence, to initially identify candidate protein coding regions in a DNA sequence .
  • For example, in a randomly generated DNA sequence with an equal percentage of each nucleotide, a stop-codon would be expected once every 21 codons.
  • If a portion of a genome has been sequenced (e.g. 5'-ATCTAAAATGGGTGCC-3'), ORFs can be located by examining each of the three possible reading frames on each strand.
  • In this sequence two out of three possible reading frames are entirely open, meaning that they do not contain a stop codon: