plement of mRNAs from the genome because alternative splicing1 is common and contributes largely to protein and functional diversity in humans and other higher organisms (Xu et al. 2011). Technologies for measuring mRNA transcripts in all their varieties, including alternatively spliced transcripts and copy-number variants, have grown rapidly in the last few years. For example, a new approach called the Glue Grant Human Transcriptome Array completes a comprehensive analysis of the human transcriptome using a 6.9 million—feature oli-gonucleotide array. The array assesses gene-level and exon-level expression by using high-density tiling of probes that cover a large collection of transcriptome. It can also detect alternative splicing and can analyze noncoding transcripts and common variants (such as single nucleotide polymorphisms) of genes (so called cSNPs) (Xu et al. 2011). This technology was recently used in a multicenter clinical program that produced high-quality reproducible data (Xu et al. 2011). It is an example of the rapid change in technologies in the -omics world and will increasingly provide new approaches to understanding how environmental factors influence the development of common diseases. Such technologies will also have many applications in the fields of microbial genomics, evolutionary biology, and other areas of interest to EPA.
• The initial preparation of the DNA sample is more labor intensive for NGS than for Sanger, but the amount of sequence data obtained per sample is substantially more.
• The number of sequencing reads from a single instrument per run is of the order of thousands with Sanger, but millions to billions with NGS; for example, a bacterial genome can be sequenced in a single run in days using NGS, versus months using Sanger sequencing.
• Read lengths from Sanger sequencing are up to 900 [base pairs], but in NGS vary from 30 to 500 [base pairs] depending on the platform.
• DNA sequencing costs have been driven down by NGS and base pair per dollar costs show a consistent 19-months doubling time reduction for Sanger sequencing. For NGS, the equivalent figure is approximately 5-months doubling time cost reduction.
• NGS can detect somatic mutations at [less than or equal to] 1%, whereas Sanger sequencing has significantly less sensitivity.
• The greater versatility of NGS is illustrated in generating whole-genome datasets, such as miRNA and ChIP-Seq; Sanger sequencing lacks this capability.
Abbreviations: ChIP-Seq, chromatin immunoprecipitation sequencing; miRNA, micro RNA; NGS, next-generation sequencing. Source: Woollard et al. 2011.
1 Alternative splicing “the process by which individual exons of pre-mRNAs are spliced to produce different isoforms of mRNA transcripts from the same gene” (Xu et al. 2011).