|Martin Smith||James Ferguson||Kirston Barton||Shaun Carswell|
The Genomic Technologies Group extends and complements the Garvan Institute's capabilities in genomic, transcriptomic, and epigenomic research by evaluating new technologies for clinical and research applications.
As genome sequencing technologies continue to advance and diversify, it is essential to constantly evaluate, implement, and develop the most informative and efficient genomic data generation methodologies. Harnessing the capabilities of the latest technologies allows researchers to answer questions with greater ease, explore new areas of biology, and develop more effective diagnostic tests.
At the moment, routinely used DNA sequencers generate extreme amounts of data to achieve accurate genome reconstruction, yet still struggle or fail to resolve important genomic features implicated in disease aetiology, such as haplotype estimations, large structural variations, epigenetic marks, copy number variations, and characterisation of repetitive elements, which make up more than 40% of the human genome.
The latest generation of sequencing technologies, such as Oxford Nanopore Technologies and 10X Genomics, facilitate many beneficial applications for genomic research. These include the following:
- Ultra long sequencing reads (>900,000 bases);
- Single cell, single molecule sequencing;
- Direct and automatic detection of epigenetic modifications (e.g., 5-methylcytosine);
- Direct RNA sequencing without conversion to DNA;
- High portability;
- Rapid turnaround of results; and
- Real-time, interactive, and dynamic acquisition.
The Genomic Technologies program is developing methods and applications involving the latest genome sequencing technologies, with a strong focus on bioinformatics, to complement existing high-throughput sequencing capabilities and maintain the Garvan Institute’s position as a global leader in genomics research. In addition to its core research capacity, the program also provides a range of application specific sequencing solutions that integrate the latest genomic sequencing platforms.
The KCCG Genomic Technologies program aims to ensure that the Garvan Institute is at the cutting edge of the rapidly evolving genomics technology landscape by evaluating new sequencing platforms, developing new methodologies, and translating these applications into clinical and commercial settings.
Nanopore sequencing service
With the recent acquisition of an Oxford Nanopore GridION sequencer, KCCG will be offering fee for service nanopore sequencing and analysis to researchers across the world in collaboration with the Garvan-Weizmann Centre for Cellular Genomics. In the future, our nanopore sequencing facility will be capable of highly scalable and affordable DNA and RNA sequencing data production thanks to our participation in the PromethION early access program. In addition to the commercial sequencing service, the Genomic Technologies program can assist internal and external collaborators in developing new sequencing protocols, applications, and data analysis in a collaborative manner.
Real-time genomic analyses
Unlike most other platforms, nanopore sequencing generates sequencing data in real time. Upon reception of purified DNA, a sample can be prepared, loaded onto a sequencer, and produce sequencing reads within 10 minutes. This capability has the potential to drastically reduce the turnaround time for DNA and RNA sequencing from several weeks to a few hours. We are exploring how real-time sequencing can provide faster and more accurate genomic diagnoses and developing the software to achieve this.
Targeted long-read sequencing
Sequencing an entire genome is unnecessary when investigating the features of a single (or a handful) of genes of interest. In such cases, a targeted sequence strategy is usually more productive and cost-effective. We are investigating approaches to efficiently enrich or select for specific genomic targets, thus providing higher sequencing resolution, improved focus, less spurious sequences, and lower cost per sample. In particular, we are focusing on methods that can leverage the information contained within long, single-molecule reads.
Single cell, single molecule sequencing
Single cell sequencing offers unprecedented resolution of gene activity throughout normal tissues and tumours. However, most single cell methodologies are limited by only being able to sequence the extremity of genes, thus lacking information about the upstream sequence. We are developing and applying new software for the analysis of long-read sequencing data from single cells, which can elucidate the entire sequence of individual gene transcripts instead of a small fragment. The higher sequence error rate of nanopore sequencing (compared to short read data), hinders the effective identification of cell barcodes (i.e., molecular tags) in single cell data. Barcodes are required to separate sequence data into the individual cells they originate from. Therefore, much information is lost using classical sequence-based approaches. By developing algorithms to analyze the raw signal instead of the processed sequence data, we can deconvolute single-cell barcodes with greater accuracy, enabling unparalleled views of the molecular inhabitants of individual cells.
Nanopore sequencing can read native RNA molecules from the cell, without prior conversion to cDNA. Until recently, RNA transcripts had to be converted to DNA using specialised enzymes. This process introduces artifacts and effectively strips away epigenetic information. Direct RNA sequencing can identify these artifacts and, in addition, identify the presence of non-standard RNA bases. We are developing methodologies to identify modified bases in biological samples, which will shed light onto the extent of epitranscriptomic features in normal health and disease.
Epigenetic processes, such as DNA methylation, are used by our cells to regulate gene expression. Techniques to identify DNA methylation are generally laborious and costly. However, epigenetic marks can be detected with nanopore sequencing when native DNA molecules are sequenced. The Genomic Technologies program is exploring how to incorporate DNA methylation signals from nanopore sequencing into clinical genomics pipelines.
New sequencing technologies can increase the speed and lower the cost of traditional genetic tests, such as BRCA1/2 testing.
Long-range variant phasing
Identifying genetic mutations is now a well-established procedure at KCCG and Genome.One, but linking distant mutations is difficult as short read sequencing provides no long-range information. Using long reads, we can identify whether distant mutations are located on the same DNA molecule, which can help determine if they have a combined or independent effect on the affected gene.
24h/rapid genomic diagnostics
Next-day whole genome sequencing reports are no longer restricted to science fiction; with the real-time sequencing platforms at KCCG, we are striving to drastically reduce the turnaround time for whole genome-derived diagnostics.
Synthetic spike-in standards (Sequins)
Dr Tim Mercer, Transcriptome Research
Prof John Mattick, RNA Biology & PlasticityDr Eva Novoa RNA, Biology & Plasticity
Prof David Thomas, Genomic Cancer Medicine
Prof Vanessa Hayes, Human Comparative & Prostate Cancer Genomics
Ruth Lyons, Human Comparative & Prostate Cancer Genomics
Dr Eva Chan, Human Comparative & Prostate Cancer Genomics
Somatic sequence variation
Prof David Thomas, Genomic Cancer Medicine
Dr Arcadi Cipponi, Genomic Cancer Medicine
Single-cell, single-molecule sequencing
Prof Chris Goodnow, ImmunogenomicsDr Mandeep Singh, ImmunogenomicsA/Prof Alex Swarbrick, Tumour ProgressionGhamdan Al Eryani, Tumour Progression
GWCCG Single Cell
Rob Salomon, GWCCG
Dr Igor Ulitsky
Oxford Nanopore Technologies
Prof Chris Mason