Biological Computing User Stories

Reginald and Nelle

Reginald Random is a post-doc who is just leaving to take up a new post and is passing his data on to a new grad student colleague, Nelle Newbien. The data are pair-end sequence reads. Each sequencing run has about 10 million reads that have Reginald has processed and split into left and right read files. He has not labelled individual paired reads uniquely but they are labelled with a unique ID for sequencing run and the left and right files are labelled in a system that was very clear to him at the time. He always labelled the left files with an "l" prefix and the right files with an "r" prefix but did not use the same root file name for the paired files.

The software for aligning the paired-end reads to the reference genome, a task which Nelle has been left to perform, expects the files to be fed in a particular order: the left-read file should be followed by the right-read file and that the order of mate pairs is the same in both files.

He had been planning to create an detailed index that explains the nomeclature of all the files before he left the data to Nelle but he was so excited about his tenure-track position that he never got round to writing the file. The data represent a substantial investment and Nelle want to be sure she is analysing the paired-end read data correctly. She gets in touch with Reginald and he promises to email her the index file soon.

Reginald's soon-to-be former advisor was very concerned that Nelle could not readily decipher the cryptic file nomenclature and strongly recommends that Reginald and Nelle attend the Software Carpentry bootcamp before he leaves the lab, or he may have to re-evaluate Reginald's authorship on the paper.

During the Software Carpentry bootcamp Reginald will learn to use a more rational and unambigious file nomenclature and how to provide metadata in a standardized format which will then allow Nelle (or anyone else) who inherits the data to understand the data structure and quickly get up to speed working with the dataset. In the unlikely event that Reginald's meta data are not correct, Nelle will also learn how to work with the files in a shell (as, due to the size of the files, the whole dataset is stored on a university server and cannot be moved on a local machine). She will also learn how to write Python scripts to perform pairwise comparisons of the files containing the data which will allow (left and right files will have the same number of lines). Nelle will gain enough programming skills to write a script that will control for the unlikely case that two pairs will actually have the same number of lines (so 4 files having the same number of lines). The script will then compare the name of the sequence in the file and pair up the correct two files.

Emma

Emma Poisson is a grad student working on mating behavior in fish. Her lab has been tracking matings between fish with many different characteristics, including things like fin shape, ventral color, and nose length. Many different technicians have collected this data over the years in a lab notebook, for many hundred (but not thousands) of pairings. Emma has hand-entered this data into an Excel spreadsheet but is getting confused by a number of things: different nomenclature for the same feature, too many traits (columns) and rows (pairings) in her spreadsheet. Moreover, because the data is largely hand-entered there are a lot of errors in it. Finally, the traits that were recorded are not terribly granular and so sometimes multiple traits can be found in a single column.

Now Emma and her advisor are trying to find correlations between mating behavior and features, but cannot figure out how to get Excel to do it and are stuck doing hand-analysis of subsets of the data in an attempt to find correlations. They are largely developing hypotheses one by one and testing them on the data set as they can. This works well for testing previously observed hypotheses but does not scale well to finding unknown correlations.

Emma attends a SWC bootcamp, learns some R, and reads this paper on how to make your data amenable to analysis. She spends a few days hand-editing the data in Excel so that it can be read into R cleanly, and then she learns how to do data aggregation, including subsetting by a specific characteristic or combination of characteristics, and summarizing these aggregates numerically and graphically. (Tools mentioned here include plyr and ggplot2.)

The effect is that Emma can now do exploratory data analysis much more quickly and comprehensively. One specific outcome is that she found that one technician has been systematically underscoring nose length in the fish. More generally she can now find interesting correlations without worrying about missing something that they didn't think of up front.

Dave

Oceanography graduate student Dave Diverseaty works with a varied (diverse) set of data collected by himself, his colleagues, and other researchers and agencies in the field. He has some training in tools (data collection, Excel, basic stats) from his undergraduate degree but further skills in this area are not explicitly taught, rather osmosed from other students and on-demand advice from advisor. In a typical project, Dave spends two weeks on an oceanographic research vessel collecting discrete and continuous water samples from many instruments. Some data are recorded manually in his lab notebook, others are output by instruments analyzing samples.

To analyze this data, Dave begins by transcribing his measurements into Excel spreadsheets and downloading data spreadsheets from external sources. To combine data from different spreadsheets, he sorts both spreadsheets by some common column (e.g., the "station" and "depth" at which they were collected) and manually copies and pastes matching records into a third combined spreadsheet. He then exports the combined spreadsheet to a CSV, loads the CSV into Ocean Data View for analysis, and during the loading process he manually labels what oceanographic variable each column maps to. He uses ODV to produce some domain-specific visualizations and, having identified a few key variables, he exports certain parts of the data for statistical analysis in R.

The statistical analysis in R reveals some errors in the original data, and Dave realizes he downloaded the wrong version of an external dataset. With extensive manual effort, Dave re-runs all the processing steps performed so far and finally arrives at a publishable result. Six months later, Dave's paper is accepted and, while in the middle of a job search, Dave has to "publish" his data. Since he's so busy, he creates a zipfile of all the Excel spreadsheets he has and posts it on his website. Six more months later, when another researcher contacts him with difficulty understanding one how one of his integrated Excel spreadsheets was produced, Dave replies that he can't remember exactly what steps he used and has moved onto a new project at OceanCorp where his job doesn't allow him time for academic research.

Current workflow:

• start with pen and paper, transcribe to Excel spreadsheets
• import external databases (tables) as Excel spreadsheets
• integrate data from different tables, usually by sorting both spreadsheets on column, manually copy and paste matches into a new spreadsheet
• plot a vs b, or a/b, ...
• find an interesting plot
• export integrated data to Ocean Data View (manually connecting columns to science variables)
• export integrated data to R
• Various statistical analyses
• Produce "professional-looking" plots
• publish?
  • Excel spreadsheets get uploaded to mandatory NSF database
  • photos of lab notebooks sometimes

Issues:

• Can't remember how I did this
• Manual steps take a long time, can be error-prone
• Redo manual effort when "new version" of data arrives
  • better cleaning
  • update external database
• High overhead of "making presentable" for publication
• Lots of context switching (between apps/systems/tools)
  • Transcription, tweaking, cleanup, integration, aggregation: Excel
  • Visualization: ODV
  • Stats: R
  • Publication-quality: ODV or R, sometimes with hand-annotations in Illustrator

What we want:

• External datasets "already in" format/system needed to do your work
• Automated
• Self-documenting/provenance (how I did it)
• Replayable/Updatable automatically
• Fewer context switches
• Interface with external tools directly, (as opposed to: copying around, in/out of files)

We want Dave to be able to work in a way that the natural progression of his work is: automated, easy-to-adapt, self-documenting, minimizes the number of times that data files are manually copied or processed, and makes it trivial to reproduce and inspect every step of the processing and analysis starting with the raw data and including all cleaning, integration, visualization, and statistics. Two aspects of a good solution will include:

1. Relational database technology, i.e., SQL—perhaps authored with the help of a GUI. Each of the data processing, integration, and aggregation steps performed in Excel, as well as automated format conversion for importing data into ODV, is represented as one SQL query deriving a new dataset from prior data.
2. Using a collaborative web service to perform all these steps so that integrating others' datasets is trivial (because they're already there), publishing is as simple as clicking a button to make the entire workflow public, and external applications can access the data directly via a web interface rather then passing around files.

Sam

Sam is a PhD student in plant biology studying the genome of several species of sunflowers. Sam is in her third year and has finished gathering together data sets from within the lab and from external collaborators. There is no bioinformatician available to help, and Sam's advisor, Eliza S. Meany, expects Sam to organize, manage, analyse, and interpret these data by next month. Sam is panicking because the data are in many different forms and she doesn't even know where to start. She has never opened a terminal and is terrified of the command line. Moreover, Sam's statistical knowledge is limited to the most basic statistics learned in an introductory math course.

At a conference, Eliza meets a colleague who tells her about a training course that will help Sam. She returns from the conference to find Sam, brow sweating and fingers cramped, working diligently to cut-and-paste the data into a single Excel spread sheet. As Eliza watches in horror, Sam's computer crashes as the Excel sheet exceeds the maximum allowed characters. "Sign up for this course," she says. Sam picks up the phone.

Software Carpentry will teach Sam to:

1. Organize the data;
2. Integrate the data and link it to available metadata;
3. Recognize the limitations of data integration;
4. Annotate a work plan so Meany's other students and collaborators can understand the process;
5. Be able to automate obvious processes that would be a waste of time to do manually; and
6. Provide a common language that allows Sam to interact effectively with bioinformaticians when future questions undoubtedly arise.

Example tasks Sam needs to do:

1. Experimental design (what happens in data driven science), controls, pre-processing (if genomes: assembly), post-processing...(e.g so many mappers, so many tools which one, which one???)
2. Resource management (CPU, storage needs, backup, compression options, etc)
3. Manipulation of the retrieved sequences;
4. Analysis of sequences by retrieving the conserved regions and identifying SNPs;
5. Visualize synteny and discover regions missing in one genome compared to others; and
6. Identify "definitive" gene list for the genomes of my favorite organism, and compare it with a subset of other genes from other studies (eg our RNAseq).
7. Workflows and use of analysis tool suites (e.g. TAVERNA, Galaxy).

Sally

Sally Species-Distribution has site observations for 15 years for endangered woodpeckers. She wants to conduct statistical analysis and map species distribution and richness for several species. Her current practice is to visually inspect and manually adjust the columns in her spreadsheets so that they are consistent across year. She then imports these manually to R and to her GIS and uses the GUI to create maps of each year and animate them.

After her training, she will be able to automate the comparison and standardization of the spreadsheets, store the information in a relational database, connect the database to R, and automate the statistical analysis and mapping of the data. Specifically, she will be able to:

• design her database schema
• write code to test data validity of the spreadsheets and import them into SQL.
• install necessary packages and write an R script to bring the data in from SQL, run statistical analyses, and make maps and graphs.

Francesco

Francesco Flotsam is head of department in a new collaborative centre for marine biology. His group is doing metagenomic studies of extreme marine environments but his department has just secured industrial funding to look for lipases that work at extremely low temperatures for new laundry products. "We're about to scale up our sequencing efforts severalfold," he explains. "We need to put some pipelines in place that will enable us to make sense of all the data that we're generating. The extremophile project is a collaborative effort, and we want to be able to share information with our research network."

Workflow:

1. collect samples - give unique identifiers with associated metadata
2. exome sequencing yields raw sequence data per sample
3. functional characterization - to pull out gene products that look like they encode enzymes - lipase (greasy stains) - many per sample
4. express genes - high throughput screening to confirm whether it does in fact encode a lipase
5. per enzyme then run an assay to then evaluate utility at low temperatures
   • substrate disappearing over time at various temperatures
   • automate the analysis of enzymatic rates across enzymes and temperatures

Computational algorithms needed:

• database schema for all labs—to relate all produced data
• use existing pipelines to generate sequence data
• sequence data shared, compiled, and version controlled among the labs;
• scan sequence data against existing database - focused chunk of InterPro - families and functional domains/motifs - identify motif that would be an active site for a lipase
• algorithm to prioritize identified gene products - those that are known to be connected to lipase activity
• linked to high throughput screening;
• assign them to the different labs for the assay

Pat

Pat is a traditional field ecologist who has amassed a large number of traditional data sets on wetland ecology, specifically ephemeral wetlands and the impact on amphibian diversity and distribution. She has started publishing in journals that require the data be made public through a repository such as Dryad. She has also realized that formatting data for sharing is much more involved than just mentally keeping track of all the accumulating spreadsheets over the years.

As Pat has started sharing data, downloading other relevant data sets and looking at patterns at a larger scale has become more appealing. But she doesn't know how to work in any other format than Excel and also many of the relevant data attributes are only in some files and not others.

Pat is starting to see that if these multiple data sets could be merged together, they would cover a substantially larger region than Pat's traditional field research occurred over and also now covers a much longer timespan as well. Analysis will certainly entail more complicated models and possibly parsing out data for different analytical processes at different spatio-temporal scales, but since Pat does everything manually, these tasks are starting to seem overwhelming. Should Pat should go back to traditional field approaches and just keep working with that data exclusively? Or is there a quick way to get up to speed on the basics of data integration, manipulation and analytical workflows?