To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS).
However, one way of avoiding this might be to use the Helicos next-generation single-molecule sequencing platform, which can generate a sequencing library from 50 pg of starting material without requiring amplification [ 4].
This single-molecule sequencing platform was successfully used for both chloroplast and microbial genomes [ 36, 37], suggesting it may be useful for solving Saccharomyces genomes for a small number of strains.
We have chosen the single-molecule sequencing (SMS) platform for RNA analysis due to its superior performance and reproducibility for quantification of RNA expression, in part due to the fact that it does not depend on PCR amplification and ligation for the library preparation [ 16].
However, the Pacific Biosciences single molecule, real-time sequencing platform holds great promise in reducing input amounts and generating sufficiently long sequences that bypass the need for large insert paired libraries.
The result is the most complete filarial nematode assembly produced thus far and demonstrates the utility of single molecule sequencing on the Pacific Biosciences platform for genetically heterogeneous metazoan genomes.
The new generation of single molecule sequencing technologies, such as the RS platform from Pacific Biosciences with longer reads and different error profiles, have brought their own QC challenges.
A second set of platforms, described as 'single molecule sequencing' (SMS) [18] by Helicos and Pacific Biosciences, eliminate the amplification steps involved in the sample preparation and sequencing process and thus profess to provide a more accurate view of the transcriptome.
Novel sequencing platforms, such as SMRT (Single Molecule Real Time sequencing) PacBio RS family [ 21] by Pacific Biosciences, generate reads with a mean length of 8.500 bases and longest reads exceeding 30 Kbp.
Pacific Biosciences technology provides a fundamentally new data type that provides the potential to overcome some limitations of current next generation sequencing platforms by providing significantly longer reads, single molecule sequencing, low composition bias and an error profile that is orthogonal to other platforms.
