Emerging technologies, such as single molecule sequencing and smaller single lab devices are not widely used yet, and Sanger sequencing of large-insert libraries is still significant [ 11].
At present de novo genome sequencing of tetraploid potato cultivars is still a challenge, however, the development of novel approaches, such as long-read single molecule sequencing and optical mapping, may change this outlook and enable the complete dissection of structural variation in heterozygous polyploid genomes in the near future [ 61, 62].
Currently, the method provides a means for the simultaneous sequencing, generation of single molecule sequences, and assignment of short (∼120 bp) from homologous PCR products obtained from multiple individuals.
We have confirmed the identity of our phased allele pairs by single-molecule sequencing and have demonstrated, in principle, that three-allele phasing (using three colors) is a potential extension to this method.
Our PCR validation results suggest that using amplification to confirm transcripts exclusively detected by single-molecule sequencing (and missed by AS sequencing) is not ideal, since any sequence that is difficult to amplify will be hard to detect using AS RNA-Seq and hard to validate using an amplification-based system.
This method broadens the application of nanopore-based single-molecule sequencing and makes it a promising and versatile tool for rapid clinical and research applications.
Single-molecule sequencing and Long Fragment read (LFR) technologies could offer a definitive haplotype on a genome-wide scale, in particular those methods that offer the possibility of long read lengths and the phasing of heterozygous SNPs into long haplotype contigs [ 25- 28].
Longer reads, greater sequencing depth, the random nature of single molecule sequencing errors and its cost and assembly performance suggests this technology will be increasingly used to produce finished microbial genomes (Koren et al., 2013).
Different hybrid assembly protocols have been developed to overcome the high error rates associated with the single molecule sequencing technology and limitations of short-read technologies (Bashir et al., 2012; English et al., 2012; Koren et al., 2012; Ribeiro et al., 2012).
10.7554/eLife.10565.015 Figure 5. Schematic of transposon-specific flanking sequence exponential anchored polymerase chain reaction amplification (FLEA-PCR) and massively parallel single molecule sequencing assay for mapping and sequencing transposon insertions.
'Third-generation', single molecule sequencing platforms are forthcoming and are reviewed in detail elsewhere [ 129].
