This blog post was written by Cristina Pomilla, Deepti Gurdasani, Martin Pollard and Tommy Carstensen
The advent of low-cost sequencing has provided a deeper understanding of the role human genetic variation plays in health and disease susceptibility. However, due to the limitations of short-read technologies, it is still very difficult to reconstruct exact DNA sequences (i.e. maternal/paternal haplotypes) or to identify large structural variants and complex genomic elements, which may all be critical to uncover the link between diseases and underlying genotypes.
SMRT® (Single Molecule Real-Time) sequencing is a ‘third generation sequencing’ technology, developed by Pacific Biosciences (PacBio), which enables us to observe the DNA polymerase/template complex as a strand of DNA is synthesised. Through delivery of very long sequence reads (over 10Kb) and circular reading around a DNA molecule, so that a single molecule is read several times, this technology allows us to better understand the structure and function of genes by accurately delineating large regions of the genome.
The SMRTLeiden Meeting, jointly organised by PacBio and Leiden University Medical Centre (6-8th June 2016) brought together experts in the field from across the globe to discuss the applications and benefits of SMRT® PacBio sequencing.
The meeting showcased various advances in the use of PacBio sequencing for the ‘assembly’ of genomes, whereby individual sequences, with overlap, can be pieced together without any previous information (de novo). Even highly curated reference genomes can benefit from this technology; re-sequencing of the human genome has helped to identify structural variation and to close many gaps in the sequence. PacBio has also been successfully used to sequence RNA, with long reads improving the ability to identify novel splicing isoforms, novel exons and transcripts. Splicing mutations and levels of alternative spliced isoforms have been associated with a variety of diseases; however, tools for reconstructing complete isoforms from short-read data have so far been inadequate.
The PacBio platform has also been shown to have clinical applications. The Anthony Nolan Trust, the largest registry of bone marrow and stem cell donors in the UK, has migrated from classical Sanger sequencing to high throughput PacBio targeted sequencing of the highly complex HLA region, as high resolution allele typing can potentially improve HLA matching and transplantation success. Mount Sinai Hospital in New York uses PacBio sequencing in daily clinical practice, including for the detection of HIV drug resistance, as well as for determining the strain of the hepatitis C virus a patient may be infected with. This new technology has also been successfully applied to the study of microbiomes and the field of epigenetics, with novel DNA modifications being detected.
Unfortunately, PacBio sequencing continues to be prohibitively expensive on a large scale, something that the manufacturers hope to address with their up-and-coming Sequel platform. Other emerging technologies, such as the Oxford Nanopore, the 10x Genomics Chromium chemistry and Dovetail Genomics sequencing technologies, promise to be more affordable than current PacBio technologies when combined with Illumina platforms, and may be able to provide similar standards of data quality.
Whilst it is difficult to say who the major players will be in this field in the next five years, the future of third generation sequencing looks to be bright, and in a period of rapid development. The information we have about genome, microbiome and transcriptome sequences across species my change dramatically over the next few years, and this will have important implications for medical genetics, and translation of these technologies into clinical practice.