Sequencing Strategies

Abstract

Several strategies to sequence complete or partial genomes are available. Complete genome sequencing is usually conducted based on shotgun-sequencing strategies, where DNA is fragmented into smaller pieces which are sequenced and finally assembled into contigs to recover the genome sequence. Two different strategies using shotgun sequencing are broadly used. For hierarchical shotgun sequencing, relatively large DNA fragments are subcloned, and physical mapping is conducted to reveal the relative order of clones, which are finally sheared and sequenced. Alternatively, by whole-genome shotgun sequencing, total DNA is directly fragmented and sequenced. As eukaryote genomes are often rich in repetitive DNA, whole-genome assembly of short reads as generated by the most used NGS methods became challenging. Generation of mate-pair libraries or the use of long-read technologies increased the quality of these genome assemblies. Several methods have been developed to sequence a reduced, but consistent representation of the genome. These so-called RADseq approaches include fragmentation of genomic DNA by using restriction enzymes. Several RADseq variants are currently in use to generate high numbers of informative markers for population genetic and phylogenetic analyses. Using hybrid enrichment techniques, it is possible to capture specifically selected sequences (e.g. conserved elements, exons, mitochondria, etc.). Similar to microarrays, DNA is bound to previously synthesized short target DNA fragments. Enrichment takes place by binding biotinylated targeted DNA to streptavidin-coated magnetic beads, while nontargeted DNA is washed away. Moreover, transcriptomes can be characterized by sequencing RNA reverse transcribed into cDNA using NGS methods. RNA-Seq became a powerful tool for de novo assembly of transcriptomes, gene finding and expression analyses.