Field guide to next‐generation DNA sequencers

Abstract

The diversity of available 2(nd) and 3(rd) generation DNA sequencing platforms is increasing rapidly. Costs for these systems range from < $100,000 to more than $1,000,000, with instrument run times ranging from minutes to weeks. Extensive trade-offs exist among these platforms. I summarize the major characteristics of each commercially available platform to enable direct comparisons. In terms of cost per megabase (Mb) of sequence, the Illumina and SOLiD platforms are clearly superior (≤ $0.10/Mb vs. > $10/Mb for 454 and some Ion Torrent chips). In terms of cost per nonmultiplexed sample and instrument run time, the Pacific Biosciences and Ion Torrent platforms excel, with the 454 GS Junior and Illumina MiSeq also notable in this regard. All platforms allow multiplexing of samples, but details of library preparation, experimental design and data analysis can constrain the options. The wide range of characteristics among available platforms provides opportunities both to conduct groundbreaking studies and to waste money on scales that were previously infeasible. Thus, careful thought about the desired characteristics of these systems is warranted before purchasing or using any of them. Updated information from this guide will be maintained at: http://dna.uga.edu/ and http://tomato.biol.trinity.edu/blog/.

Advances in genomics for flatfish aquaculture

“…Thereafter, the clusters of DNA templates are sequenced by synthesis or ligation in a phased approach. There are three main SGS platforms (reviewed by Glenn 2011;Metzker 2010;Voelkerding et al 2009): (a) the 454 platform (Roche Applied Science), based on emulsion PCR followed by pyrosequencing reactions to produce approximately 0.4-0.5 gigabases (Gb) per run rendering sequences of 300-400 base pairs (bp) mean length (although a new chemistry is ongoing to reach 600-700 bp mean length) (e.g. 454 FLX Titanium sequencer); (b) the SolexaIllumina Ò platform (Illumina, Inc.), which uses bridge PCR for DNA amplification and dye-labelled terminators in a polymerase-mediated reaction for sequencing, and produces 200-300 Gb/run with a sequence mean length of 100 bp (e.g.…”

“…An additional emerging technology is the ion torrent, considered as an intermediate approach between the SGS and TGS because it does not require any laser, fluorescent dyes or cameras, reducing drastically the costs and generating an important volume of genomic information, although it is still dependent on a PCR amplification step (Schadt et al 2010). For more technical and methodological aspects about SGS and TGS platforms, extensive reviews describing and comparing chemical costs, read performance and some other characteristics are available (Metzker 2010;Schadt et al 2010;Glenn 2011;Pareek et al 2011;Zhang et al 2011a). …”

“…These include whole-genome sequencing, bacterial artificial chromosome (BAC) analysis and target re-sequencing, single nucleotide polymorphism (SNP) and mutation identification, transcriptome analysis (RNA-seq), small RNA profiling, analysis of epigenetic markers and chromatin structure using seq-based methods (ChIP-seq, methyl-seq and Dnase-seq) or metagenomics (Metzker 2010;Glenn 2011). Each NGS platform shows advantages or disadvantages depending on the application of interest.…”

“…In general, those platforms that produce long reads are optimal for de novo genome and transcriptome characterization since long sequences are more amenable for computational analysis and assembly, whereas those producing shorter reads are often targeted for re-sequencing, expression profiling and metagenomics. However, the continuous development of advanced NGS technologies has resulted in most of them being used for a wide range of applications (Glenn 2011). Although the application of NGS in flatfish genomics research has just started, we will summarize the main applications in which these technologies are currently being used.…”

“…Altogether, these data indicate that flatfish genomes are small and compact, which should facilitate de novo whole-genome sequencing, assembly and further characterization. However, if we consider the mean flatfish genome size indicated above, and we assume neither bias in sequencing reads nor the time required for the preparation of genomic libraries, the mean time to obtain a number of reads equivalent to a flatfish genome with a 209 coverage by using NGS would be 1-11 days (estimated mean speed of 50 Mb/h for 454 platform or *1,000 Mb/h for Illumina) (Glenn 2011). However, although a high volume of genomic information can be generated in a short time with NGS methods, the strategy followed for the construction of the genomic libraries, sequence read length and quality, and genome structure (repetitive sequences, gene duplications, etc.)…”