Clinical applications of sequencing take center stage

With the HiSeq and the MiSeq platforms, Illumina is now firmly established as the most widely used sequencing technology. Geoff Smith (Illumina, USA) announced the recent acquisition of Moleculo, a new protocol extending contiguous sequence length. Jonathan Rothberg (Life Technologies, USA) reported on the 100-fold increases in sequencing output in just two years on the Ion Torrent and Proton platforms, stating they are 'not seeing the end of capacity' for the semiconductor-based technology. In addition, Jonas Korlach (Pacific Biosciences, USA) presented impressive results on particularly difficult sequences and showed that pathogen genomes could be quickly finished using the SMRT platform's long reads. On the other hand, James Knight (Roche, USA) discussed the detailed sequencing of RP11 - the individual who contributed 70% of the reference genome - closing many reference gaps and leading to the identification of reference errors.

The vast accumulation of genetic data, the use of combinations of sequencing technologies and a variety of algorithmic improvements have led to the identification of certain limitations and recurring error types of current technologies. Mark DePristo (Broad Institute, USA) described the observation of 'fake SNPs' that arise from misassembling reads around short gaps, and discussed how these were resolved using the HaplotypeCaller software. He also emphasized that PCR amplification in the library preparation step can be a major source of error. Meanwhile, Jay Shendure (University of Washington, USA) described an updated version of molecular inversion probes that is highly multiplexed and rebalanced for solving problems caused by extremes in GC content; he also reported finding many de novo mutations that were missed by exome sequencing. In addition, I reported observing thousands of genomic segments enriched in false-positive findings, based on analysis of hundreds of complete genomes (Gustavo Glusman, Institute for Systems Biology, USA).

Exome sequencing is prevalent: more than 100,000 exomes have now been reported. Several presenters discussed limitations in exome sequencing, and contrasted results with whole genome sequencing (WGS). Stephen Scherer (Hospital for Sick Children, Canada) showed that approximately 13% of exons are absent from exome capture arrays, and that WGS data yield more uniform coverage of the exome. He further reported that 6 out of 56 clinically relevant variants observed in a WGS study of autism would not have been captured by exome sequencing. Interestingly, Malachi Griffith (Washington University School of Medicine, USA) showed examples of short intronic deletions, which are invisible to exome analysis, and demonstrated how these can affect gene expression levels. Michael Talkowski (Harvard Medical School, USA) stressed the difficulty in identifying copy-number variants and aneuploidy from exome data. Overall, it seems there is a clear trend for WGS becoming the ultimate diagnostic tool.

The central theme of this year's AGBT was the application of sequencing technologies to clinical needs. Recurring topics of discussion included the need for speed in processing pipelines (from samples to interpretation), validation of results and clinical reporting, particularly for incidental findings.

Beyond the clinic, a dizzying diversity of applications was presented. Kjersti Aagaard (Baylor College of Medicine, USA) described revelations of detailed metagenomic sequencing applied to obstetrics: her team found that the composition of the vaginal microbiome shifts during pregnancy, and that the placenta has a nonpathogenic commensal microbiome most similar in composition to that of subgingival plaques. Ross Hardison (Pennsylvania State University, USA) reported enrichment of gene-regulating variants outside coding regions, and modeling gene expression based on integration of epigenetic features, multi-species alignments and transcription factor motif enrichment. Eric Schadt (Pacific Biosciences, USA) highlighted massive copy number variation impact on gene expression levels. Leonid Moroz (University of Florida, USA) studied memory and epigenetic modifications in giant snail neurons, and found massive DNA demethylation in quick response to neurotransmitters. He reported being able to perform sequencing on an expedition ship, simplifying oceanic exploration. Leonard Lipovich (Wayne State University, USA) reported thousands of novel long non-coding RNAs. Chia-Lin Wei (Joint Genome Institute, USA) presented a detailed and riveting connectivity map of chromatin interactions between promoters and enhancers, within and between chromosomes. Finally, Mark Yandell (University of Utah, USA) discussed applying 'pigeonomics' to the same pigeon species that Darwin studied.

As attention shifts from sequencing to extracting meaning from the data, there is a proliferation of commercial analytical tools - frequently cloud-based solutions - from companies such as Ingenuity Systems, Agilent, Maverix Biomics, DNAnexus and Omicia.

There was a modest crop of novel algorithms for computational analysis of sequence data. Andrew Farrell (Boston College, USA) presented RUFUS, an ingenious algorithm for detecting differences between two samples that requires no reference sequence. RUFUS promises to have many applications, from cancer to RNA editing. Aaron Quinlan (University of Virginia, USA) reported on the development of LUMPY, a probabilistic framework integrating diverse signals for structural variant discovery.

Despite the wealth of data described at the meeting, one important issue remains unresolved: though vast numbers of exomes and whole genomes have been obtained, and some progress was reported in databasing and sharing of medical information and treatment outcomes, much work remains to be done to make these data available to the research community.