Ion-Torrent. The bead libraries derived from emulsion PCR are deposited into a “chip” with millions of cavities or wells that fit only one well. Each well is part of a microtransistor that integrates the chip, where voltage changes can be registered individually. Sequencing proceeds in a similar fashion as pyrosequencing, but instead of light emission, the addition of each base produces a voltage change. Modified form Niedringhaus et al. (2011). 

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... calibration for long stretches of a single nucleotide has its limits, and it is usually inaccurate when > 6 bases of the same nucleotide are added in a row (Mardis, 2008). Although in the last years the length of reads obtained by pyrosequencing has increased considerably (Shokralla et al., 2012), the coverage for this approach is still the lowest for the currently available platforms, ranging from 7-10X, depending on genome size (i.e. Barbazuk et al., 2007; Wheeler et al., 2008; Vera et al., 2008). Illumina genome analyzer (GAIIx) . This is one of the most praised massive sequencing platforms, due to its high quality sequences and great coverage (millions of simultaneously sequenced fragments). The procedure for library construction is also based on synthesis of complementary strands of DNA through PCR. However, the specifics of such synthesis have notable differences with the emulsion PCR used by 454. As most current massive sequencing protocols, DNA fragmentation and size selection are necessary steps prior to library construction. Once fragments of a desired size are selected, platform- specific adaptors are added which allows attachment to the sequencing matrix, a flow-cell device that will support bridge amplification and sequencing per se . On this matrix, each attached fragment is amplified producing multiple and identical DNA copies in a cluster (Fig. 2). Illumina ́s matrix has 8 separate channels (or lines) in each of which a library of clusters can be created. Each cluster is sequenced by synthesis, in which all 4 nucleotides are added simultaneously to the flow device, along with DNA polymerase for addition into the oligo-primed fragments that form the clusters. For this sequencing approach, the addition of a nucleotide blocks subsequent incorporation of nucleotides, interrupting the synthesis, and releases a fluorescent signal that is unique for each type of nucleotide. Imaging of the fluorescent signal follows the incorporation. After imaging, the blocking group is removed and leaves DNA strands ready for the next nucleotide incorporation by DNA polymerase. This series of steps continues for a specific number of rounds allowing read-lengths of 60-150 bases (Glenn, 2011). Applied biosystems SOLiD TM sequencer . Sequencing by oligo ligation detection (SOLiD). This platform has the best quality of sequences and the smallest error rate (Table 1), as a result of the ligation-based approach. The library construction occurs through an emulsion PCR with small magnetic beads (similar to 454/Roche). After completion of library construction, the resulting beads are attached covalently to a flow-cell glass slide. Compared to other platforms, SOLiD has a major difference in that the approach taken for sequencing the amplified fragments, uses DNA ligase as illustrated in figure 3. The slide with attached beads is exposed to cyclic enzymatic reactions. During the first cycle a primer sequence is incorporated, that is complementary to the adaptor ligated to the fragments as well as a ligase and 4 fluorescently labeled 2- base encoded probes. Non-ligated probes are then washed away, followed by the imaging of the released fluorescence that identifies the ligated probe (Landergren et al., 1988). The cycle is repeated to remove the fluorescent dye and regenerate the 5 ́-PO4 groups for 10 subsequent ligation cycles (Fig. 3). A second ligation round is performed with a “n-1” primer, which resets the interrogated sequence one base downstream, and then the 10 cycle ligation proceeds. Four more rounds of ligation cycles are performed with progressively “n-1” primers. Color calls from the 5-ligation rounds are then ordered into a linear sequence (the color space) and compared to a linear sequence to decode DNA problem sequence. The read length of SOLiD was initially 35 bp with a final output of 3 Gb per run (Liu et al., 2012). These numbers have increased up to 75 bp per read and 10 Gb per run (Shokralla et al., 2012), with high accuracy in the assignment of bases due to the 2-base sequencing method (accuracy of 99.85% after filtering; Liu et al., 2012; Table 1). Ion-torrent ( the chip is the machine ). This platform is gaining popularity in the market mainly due to the low cost of sequencing runs and the possibility of in-house daily use without many technical requirements or maintenance. The library construction procedure is almost identical to 454 or pyrosequencing, including DNA fragmentation and adaptor ligation. The bead library from the emulsion PCR is deposited into a “chip” with millions of wells (165- 600 million depending on the specific version; Shokralla et al., 2012), where only 1 bead fits in each well. The extraordinary aspect of this technology is the chip itself. Each well is integrated to the chip’s ion-sensitive layer and a proprietary ion sensor to register the very small voltage changes (per well) that result from nucleotide addition during DNA sequencing by synthesis (Rothberg et al., 2011, Fig. 4). As in 454, nucleotide addition is not followed by termination and it proceeds in cycles, 1 nucleotide after another. This results in the same problem that the pyrosequencing platforms have with homopolymer detection. The read length of ion Torrent is currently about 150 bp with a final output of 3 Gb per run (Liu et al., 2012), but it has increased up to 75 additional bp per read, and up to 10 Gb per run (Shokralla et al., 2012; Table 1). Single molecule real time (SMRT) sequencing . Technological bets on massive sequencing are focused on the possibility to sequence single molecules in real time or “single molecule real time” (SMRT) sequencing. These technological developments are referred to as the third generation sequencing and, to date, only Pacific Biosciences (PacBio) has released a commercial platform implementing SMRT sequencing. SMRT implements the possibility of attaching a polymerase to a sequencing matrix and be able to follow in real time the synthesis process of a single DNA molecule (Fig. 5). An important feature of SMRT is that it does not require library construction as a prior step to sequencing, increasing the sequence production rate. Also, this technology allows for longer reads. The read length of Pacific Biosciences system has been reported to be up to 1 500 bp with a final output of 60- 75 Mb per run (Shokralla et al., 2012 ; Table 1). However, it is difficult to score single base additions in real time. The main problem in scoring real time single base additions is the high speed at which each polymerase synthesizes DNA exhibits stochastic fluctuations (Eid et al., 2009), thus, each enzyme has to be monitored individually and nucleotide additions registered at the appropriate speed in real time. This difficulties result in the highest error rates among NGS platforms (Table 1). Currently, the main bottlenecks in genomic studies are in the post-sequence stages or assembly, which usually requires major computational capacities (Henson et al., 2012). Genomes sequences are variable (for instance, including heterozygosity in diploid organisms) and can be highly repetitive, and during assembly it is necessary to distinguish this “real” variation from sequencing errors and stay within reasonable computational times, making assembly a complex problem (Henson et al., 2012). The task would be much simpler if it could be determined whether a given set of reads corresponds to overlapping positions on the genome. In this sense, generally longer reads help to correctly find overlapping regions. Given the presence of short reads in all NGS platforms, it is clear that assembling genomes, metagenomes or transcriptomes is a task that faces enormous challenges, which are even more challenging due to high error rates. The ease and accuracy of assembly depends on the degree of overlapping between reads. Two reads are considered as overlapping when there is a sequence match between reads that is long enough to be reliably distinguished from a random event (Henson et al., 2012). Thus, high uncertainty in assembly arises from locations in which not enough overlap is present to extend the genome sequence. In combination, coverage and read length increase confidence levels of the assembly process. Experience and models show that a good assembly using Sanger reads requires each base to be covered on average by at least 3 reads (3X, Lander and Waterman, 1988). However, for the short reads of NGS platforms, this number can rise up to 30X (Farrer et al., 2009; Meyer et al., 2012). High error rates also affect assembly and thus higher coverage is needed. Published genomes have used between 5X and 10X with Sanger (Adams et al., 2000; Venter et al., 2001; Venter et al., 2004), but new publications have begun to report 50X, 100X and higher coverage with NGS (Diguistini et al., 2009; Quail et al., 2012). However, even with high coverage, overcoming the problem of repeats and derived assembly gaps sometimes need to be spanned by paired reads (2 reads generated from a single fragment of DNA and separated by known distance), which are available for most NGS platforms (Schatz et al., 2010). Much research has been published on the assembly problem, as well as development of new assembly algorithms and platforms (for recent reviews see Schatz et al., 2010; Nagarajan and Pop, 2013). Nevertheless, it has been suggested that the best approach is to use a reference genome sequence as a guide to resolve repeats, an approach known as “comparative assembly” (Pop et al., 2004). A detailed analysis by Schatz et al. (2010) on the assembly of large genomes using NGS (Illumina and 454) showed that assemblies with these platforms are inferior than those accomplished using Sanger technology, but recognized the appeal of the lower costs of NGS. In this paper, guidelines are given to decide the best way to proceed in terms of choosing the platform and assembly method when pursuing a genome sequencing project. As we mention ...