Currently most used NGS platforms have restrictions on the length of the sequencing library molecules and read length and long nucleic acid molecules need to be fragmented prior to sequencing. Reconstruction of original molecules from shorter fragments is a complex task and at the moment cannot be solved entirely. For example, it is difficult to rebuild the full sequence of chromosomes, because of homologous and repetitive genomic regions and it is problematic to determine haplotypes. In transcriptome studies fragmentation of RNA molecules interferes with determination of structure and expression levels of splice variants. So valuable information about genome and transcriptome is being lost and the sequencing data we get is not complete.
This problem could be solved by increasing the length of sequenced fragments up to tens of kilobases. There exist already the third generation sequencing platforms, which can use significantly longer templates and generate longer reads. However they are characterized by a considerably higher price per nucleotide and lower throughput. It would be very attractive to gain complete sequencing information using NGS short-read platforms (Illumina) which are widely used, are high-throughput and cost-efficient.
The researchers from the Max-Planck-Institute for Molecular Genetics suggest an approach which allows preserving information about the content of long NA molecules despite fragmentation and mixing together fragments from different macromolecules. In general, the NA molecules are labelled at multiple sites prior to fragmentation, thus the fragments of each individual NA molecule receive identical codes or a predictable set of codes. The subsequent analysis allows identification and grouping together of fragments, which belonged to the same NA molecule before dissociation. In the NGS field this invention would mean a break-through in the de novo sequencing, phased sequencing and transcriptome sequencing applications. Inventors suggest several methodological solutions for molecular coding.
PCT application: PCT / EP2013 / 078174