DNA sequencing is a group of techniques that are used to obtain the sequence of nucleotides in DNA. When DNA sequencing is done for the entire genome of an organism then it is called genome-sequencing.
There are basically two main methodological principles for DNA sequencing:
- Sequencing by termination: Sanger sequencing
- Sequencing by synthesis: Most next generation sequencing methods
Sanger sequencing depends on termination of PCR reaction at different positions of a nucleotide. PCR termination happens because of the incorporation of a non-extensible di-deoxy-nucleotide (it lacks 3' OH; the triphosphate form is abbreviated as ddNTP), instead of a usual deoxy-nucleotide. This leads to formation of multiple products which indicate the location of a nucleotide in the DNA.
Sequencing by synthesis involves recording the nucleotide as it is inserted in the growing DNA.
Refer to the wikipedia page on DNA sequencing for basic details.
Next generation sequencing (NGS) methods use a high level of automation that enables large scale sequencing. Refer to this review for more details on NGS.
Applications and variants of DNA sequencing:
- ChIP-seq: Chromatin Immunoprecipitation followed by DNA sequencing (ChIP-seq) is used to identify regions of DNA that is bound by a certain protein, which in most cases is a transcription factor.
- Nucleosome sequencing: Sequencing the regions bound the nucleosome; used to study chromatin architecture
- Bisulphite sequencing: Bisulphite to deaminate methylated cytosine to thymine. This when compared to untreated DNA sequence, reveals sites of cytosine methylation.
- MeDIP-seq: Methylated-DNA immunoprecipitation followed by sequencing: Also used to find methylated regions of DNA but uses an antibody to precipitate methylated DNA (cytosine).
- DNAse Hypersensitive region mapping: DNA is treated with DNAse to identify regions protected by protein binding. Used for studying chromatin architecture
- HiC: Genome level implementation of chromosome conformation capture (3C). Used for studying DNA-DNA interactions.
Questions that can have this tag:
- Questions related to principle behind the sequencing techniques
- Questions related to principle behind any of the above mentioned implementations of DNA sequencing
- Questions related to experimental troubleshooting for any of these techniques
- Questions related to analysis of sequencing data