How to write a paper in four minutes

Three years ago, when my coauthors (Páll Melsted, Nicolas Bray, Harold Pimentel) and I published the “kallisto paper” on the arXiv (later Bray et al. “Near-optimal probabilistic RNA-seq quantification“, 2016), we claimed that kallisto removed a major computational bottleneck from RNA-seq analysis by virtue of being two orders of magnitude faster than other state-of-the-art quantification methods of the time, without compromising accuracy. With kallisto, computations that previously took days, could be performed as accurately in minutes. Even though the speedup was significant, its relevance was immediately questioned. Critics noted that experiments, library preparations and sequencing take at least months, if not years, and questioned the relevance of a speedup that would save only days.

One rebuttal we made to this legitimate point was that kallisto would be useful not only for rapid analysis of individual datasets, but that it would enable analyses at previously unimaginable scales. To make our point concrete, in a follow-up paper (Pimentel et al., “The Lair: a resource for exploratory analysis of published RNA-seq data”, 2016) we described a semi-automated framework for analysis of archived RNA-seq data that was possible thanks to the speed and accuracy of kallisto, and we articulated a vision for “holistic analysis of [short read archive] SRA data” that would facilitate “comparison of results across studies [by] use of the same tools to process diverse datasets.” A major challenge in realizing this vision was that although kallisto was fast enough to allow for low cost processing of all the RNA-seq in the short read archive (e.g. shortly after we published kallisto, Vivian et al., 2017 showed that kallisto reduced the cost of processing per sample from $1.30 to $0.19, and Tatlow and Piccolo, 2016 achieved $0.09 per sample with it), an analysis of experiments consists of much more than just quantification. In Pimentel et al. 2016 we struggled with how to wrangle metadata of experiments (subsequently an entire paper was written by Bernstein et al. 2017 just on this problem), how to enable users to dynamically test distinct hypotheses for studies, and how to link results to existing databases and resources. As a result, Pimentel et al. 2016 was more of a proof-of-principle than a complete resource; ultimately we were able to set up analysis of only a few dozen datasets.

Now, the group of Avi Ma’ayan at the Icahn School of Medicine at Mount Sinai has surmounted the many challenges that must be overcome to enable automated analysis of RNA-seq projects on the short read archive, and has published a tool called BioJupies (Torre et al. 2018). To assess BioJupies I began by conducting a positive control in the form of analysis of data from the “Cuffdiff2” paper, Trapnell et al. 2013. The data is archived as GSE37704. This is the dataset I used to initially test the methods of Pimentel et al. 2016 and is also the dataset underlying the Getting Started Walkthrough for sleuth. I thought, given my familiarity with it, that it would be a good test case for BioJupies.

Briefly, in Trapnell et al. 2013, Trapnell and Hendrickson performed a differential analysis of lung fibroblasts in response to an siRNA knockdown of HOXA1 which is a developmental transcription factor. Analyzing the dataset with BioJupies is as simple as typing the Gene Expression Omnibus (GEO) accession on the BioJupies searchbox. I clicked “analyze”, clicked on “+” a few times to add all the possible plots that can be generated, and this opened a window asking for a description of the samples:

I selected “Perturbation” for the HOXA1 knockdown samples and “Control” for the samples that were treated with scrambled siRNA that did not target a specific gene. Finally, I  clicked on “generate notebook”…

and…

BioJupies displayed a notebook (Trapnell et al. 2013 | BioJupies) with a complete analysis of the data. Much of the Trapnell et al. 2013 analysis was immediately evident in the notebook. For example, the following figure is Figure 5a in Trapnell et al. 2013. It is a gene set enrichment analysis (GSEA) of the knockdown:

BioJupies presents the same analysis:

It’s easy to match them up. Of course BioJupies presents a lot of other information and analysis, ranging from a useful PCA plot to an interesting L1000 connectivity map analysis (expression signatures from a large database of over 20,000 perturbations applied to various cell lines that match the signatures in the dataset).

One of the powerful applications of BioJupies is the presentation of ARCHS⁴ co-expression data. ARCHS⁴ is the kallisto computed database of expression for the whole and is the primary database that enables BioJupies. One of its features is a list of co-expressed genes (as ascertained via correlation across the whole short read archive). These are displayed in BioJupies making it possible to place the results of an experiment in the context of “global” transcriptome associations.

While the Trapnell et al. 2013 reanalysis was fun, the real power of BioJupies is clear when analyzing a dataset that has not yet been published. I examined the GEO database and found a series GSE60538 that appears to be a partial dataset from what looks like a paper in the works. The data is from an experiment designed to investigate the role of Sox5 and Sox6 in the mouse heart via two single knockout experiments, and a double knockout. The entry originates in 2014 (consistent with the single-end 50bp reads it contains), but was updated recently. There are a total of 8 samples: 4 controls and 4 from the double knockout (the single knockouts are not available yet). I could not find an associated paper, nor was one linked to on GEO, but the abstract of the paper has already been uploaded to the site. Just as I did with the Trapnell et al. 2013 dataset, I entered the accession in the BioJupies website and… four minutes later:

The abstract of the GSE60538 entry states that “We performed RNA deep sequencing in ventricles from DKO and control mice to identify potential Sox5/6 target genes and found altered expression of genes encoding regulators of calcium handling and cation transporters” and indeed, BioJupies verifies this result (see Beetz et al. GSE60538| BioJupies):

Of course, there is a lot more analysis than just this. The BioJupies page includes, in addition to basic QC and datasets statistics, the PCA analysis, a “clustergrammer” showing which genes drive similarity between samples, differentially expressed genes (with associated MA and volcano plots), gene ontology enrichment analysis, pathway enrichment analysis, transcription factor enrichment analysis, kinase enrichment analysis, microRNA enrichment analysis, and L1000 analysis. In a sense, one could say that with BioJupies, users can literally produce the analysis for a paper in four minutes via a website.

The Ma’ayan lab has been working towards BioJupies for some time. The service is essentially a combination of a number of tools, workflows and resources published previously by the lab, including:

• Elysium: RNA-Seq Alignment in the Cloud
• Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool
• KEA: kinase enrichment analysis
• LINCS Canvas Browser: interactive web app to query, browse and interrogate LINCS L1000 gene expression signatures
• Massive mining of publicly available RNA-seq data from human and mouse

With BioJupies, these tools become more than the sum of their parts. Yet while BioJupies is impressive, it is not complete. There is no isoform analysis, which is unfortunate; for example one of the key points of Trapnell et al. 2013 was how informative transcript-level analysis of RNA-seq data can be. However I see no reason why a future release of BioJupies can’t include detailed isoform analyses. Isoform quantifications are provided by kallisto and are already downloadable via ARCHS⁴. It would also be great if BioJupies were extended to organisms other than human and mouse, although some of the databases that are currently relied on are less complete in other model organisms. Still, it should even be possible to create a BioJupies for non-models. I expect the authors have thought of all of these ideas. I do have some other issues with BioJupies: e.g. the notebook should cite all the underlying programs and databases used to generate the results, and while it’s neat that there is an automatically generated methods section, it is far from complete and should include the actual calls made to the programs used so as to facilitate complete reproducibility. Then, there is my pet peeve: “library size” is not the number of reads in a sample. The number of reads sequenced is “sequencing depth”.  All of these issues can be easily fixed.

In summary, BioJupies represents an impressive breakthrough in RNA-seq analysis. It leverages a comprehensive analysis of all (human and mouse) publicly available RNA-seq data to enable rapid and detailed analyses that transcend what has been previously possible. Discoveries await.