Sunday, July 26, 2009

Researchers get hands-on practice using GeneSifter to analyze Next Gen data and SNPs at the UT-ORNL-KBRIN 2009 summit

As the recently-published meeting report attests (1), while the much of the genomics world was bemoaning the challenge of working with Next Generation DNA sequence (NGS) data, attendees at the UT-ORNL-KBRIN summit got an opportunity to play with the data first-hand. These lucky researchers, among the first in the nation to work with NGS data outside of a genome center, were participants in a hands-on education workshop presented by Dr. Sandra Porter from Digital World Biology.

This opportunity came about through a collaboration between Geospiza and Digital World Biology. Data were loaded and processed through GeneSifter’s analysis pipelines before the workshop to align data to reference sequences and perform the secondary analyses. During the workshop, Porter led researchers through typical steps in the tertiary analysis phase. Workshop participants were able to view gene lists and analyze information from both Illumina and SOLiD datasets. The analyses included: working with thumbnail graphics to see where reads map to transcripts and assess coverage, viewing the number of reads mapping to each transcript, and comparing the number of reads mapping to different genes under different conditions to investigate gene expression.

An additional workshop focused on using iFinch and FinchTV to view SNP data in chromatogram files generated by Sanger sequencing and working with structures to see how a single nucleotide change can impact the structure of a protein.

1. Eric Rouchka and Julia Krushkal. 2009. Proceedings of the Eighth Annual UT-ORNL-KBRIN Bioinformatics Summit 2009. BMC Bioinformatics 10(Suppl 7):I1doi:10.1186/1471-2105-10-S7-I1.

Sunday, July 12, 2009

Bloginar: Scalable Bioinformatics Infrastructures with BioHDF. Part V: Why HDF5?

Through the course of this BioHDF bloginar series, we have demonstrated how the HDF5 (Hierarchical Data Format) platform can successfully meet current and future data management challenges posed by Next Generation Sequencing (NGS) technologies. We now close the series by discussing the reasons why we chose HDF5.

For previous posts, see:

  1. The introduction
  2. Project background
  3. Challenges of working with NGS data
  4. HDF5 benefits for working with NGS data

Why HDF5?

As previously discussed, HDF technology is designed for working with large amounts of complex data that naturally organize into multidimensional arrays. These data are composed of discrete numeric values, strings of characters, images, documents, and other kinds of data that must be compared in different ways to extract scientific information and meaning. Software applications that work with such data must meet a variety of organization, computation, and performance requirements to support the communities of researchers where they are used.

When software developers build applications for the scientific community, they must decide between creating new file formats and software tools for working with the data, or adapting existing solutions that already meet a general set of requirements. The advantage of developing software that's specific to an application domain is that highly optimized systems can be created. However, this advantage can disappear when significant amounts of development time are needed to deal with the "low-level" functions of structuring files, indexing data, tracking bits and bytes, making the system portable across different computer architectures, and creating a basic set of tools to work with the data. Moreover, such a system would be unique, with only a small set of users and developers able to understand and share knowledge concerning its use.

The alternative to building a highly optimized domain-specific application system is to find and adapt existing technologies, with a preference for those that are widely used. Such systems benefit from the insights and perspective of many users and will often have features in place before one even realizes they are needed. If a technology has widespread adoption, there will likely be a support group and knowledge base to learn from. Finally, it is best to choose a solution that has been tested by time. Longevity is a good measure of the robustness of the various parts and tools in the system.

HDF: 20 Years in Physical Sciences

Our requirements for high-performance data management and computation system are these:

  1. Different kinds of data need to be stored and accessed.
  2. The system must be able to organize data in different ways.
  3. Data will be stored in different combinations.
  4. Visualization and computational tools will access data quickly and randomly.
  5. Data storage must be scalable, efficient, and portable across computer platforms.
  6. The data model must be self describing and accessible to software tools.
  7. Software used to work with the data must be robust, and widely used.

HDF5 is a natural fit. The file format and software libraries are used in some of the largest data management projects known to date. Because of its strengths, HDF5 is independently finding its way into other bioinformatics applications and is a good choice for developing software to support NGS.

HDF5 software provides a common infrastructure that allows different scientific communities to build specific tools and applications. Applications using HDF5 typically contain three parts: one or more HDF5 files to store data, a library of software routines to access the data, and the tools, applications and additional libraries to carry out functions that are specific to a particular domain. To implement an HDF5-based application, a data model be developed along with application specific tools such as user interfaces and unique visualizations. While implementation can be a lot of work in its own right, the tools to implement the model and provide scalable, high-performance programmatic access to the data have already been developed, debugged, and delivered through the HDF I/O (input/output) library.

In earlier posts, we presented examples where we needed to write software to parse fasta formatted sequence files and output files from alignment programs. These parsers then called routines in the HDF I/O library to add data to the HDF5 file. During the import phase, we could set different compression levels and define the chunk size to compress our data and optimize access times. In these cases, we developed a simple data model based on the alignment output from programs like BWA, Bowtie, and MapReads. Most importantly, we were able to work with NGS data from multiple platforms efficiently, with software that required weeks of development rather than the months and years that would be needed if the system was built from scratch.

While HDF5 technology is powerful "out-of-the-box," a number of features can still be added to make it better for bioinformatics applications. The BioHDF project is about making such domain-specific extensions. These are expected to include modifications to the general file format to better support variable data like DNA sequences. I/O library extensions will be created to help HDF5 "speak" bioinformatics by creating APIs (Application Programming Interfaces) that understand our data. Finally, sets of command line programs and other tools will be created to help bioinformatics groups get started quickly with using the technology.

To summarize, the HDF5 platform is well-suited for supporting NGS data management and analysis applications. Using this technology, groups will be able to make their data more portable for sharing because the data model and data storage are separated from the implementation of the model in the application system. HDF5's flexibility for the kinds of data it can store, makes it easier to integrate data from a wide variety of sources. Integrated compression utilities and data chunking make HDF5-based systems as scalable as they can be. Finally, because the HDF5 I/O library is extensive and robust, and the HDF5 tool kit includes basic command-line and GUI tools, a platform is provided that allows for rapid prototyping, and reduced development time, thus making it easier to create new approaches for NGS data management and analysis.

For more information, or if you are interested in collaborating on the BioHDF project, please feel free to contact me (todd at

Monday, July 6, 2009

Bloginar: Scalable Bioinformatics Infrastructures with BioHDF. Part IV: HDF5 Benefits

Now that we're back from Alaska and done with the 4th of July fireworks, it's time to present the next installment of our series on BioHDF.

HDF highlights
HDF technology is designed for working with large amounts of scientific data and is well suited for Next Generation Sequencing (NGS). Scientific data are characterized by very large datasets that contain discrete numeric values, images, and other data, collected over time from different samples and locations. These data naturally organize into multidimensional arrays. To obtain scientific information and knowledge, we combine these complex datasets in different ways and (or) compare them to other data using multiple computational tools. One difficulty that plagues this work is that the software applications and systems for organizing the data, comparing datasets, and visualizing the results are complicated, resource intensive, and challenging to develop. Many of the development and implementation challenges can be overcome using the HDF5 file format and software library

Previous posts have covered:
1. An introduction
2. Background of the project
3. Complexities of NGS data analysis and performance advantages offered by the HDF platform.

HDF5 changes how we approach NGS data analysis.

As previously discussed, the NGS data analysis workflow can be broken into three phases. In the first phase (primary analysis) images are converted into short strings of bases. Next, the bases, represented individually or encoded as di-bases (SOLiD), are aligned to reference sequences (secondary analysis) to create derivative data types such as contigs or annotated tables of alignments, that are further analyzed (tertiary analysis) in comparative ways. Quantitative analysis applications, like gene expression, compare the results of secondary analyses between individual samples to measure gene expression and identify mRNA isoforms, or make other observations based on a sample’s origin or treatment.

The alignment phase of the data analysis workflow creates the core information. During this phase, reads are aligned to multiple kinds of reference data to understand sample and data quality, and obtain biological information. The general approach is to align reads to sets of sequence libraries (reference data). Each library contains a set of sequences that are annotated and organized to provide specific information.

Quality control measures can be added at this point. One way to measure data quality is to ask how many reads were obtained from constructs without inserts. Aligning the read data to a set of primers (individually and joined in different ways) that were used in the experiment, allows us to measure the number reads that match and how well they match. A higher quality dataset will have a larger proportion of sequences matching our sample and a smaller proportion of sequences that only match the primers. Similarly, different biological questions can be asked using libraries constructed of sequences that have biological meaning.

Aligning reads to sequence libraries is the easy part. The challenge is analyzing the alignments. Because the read datasets in NGS assays are large, organizing alignment data into forms we can query is hard. The problem is simplified by setting up multistage alignment processes as a set of filters. That is, reads that match one library are excluded from the next alignment. Differential questions are then asked by counting the numbers of reads that match each library. With this approach, each set of alignments is independent of the other alignments and a program only needs to analyze one set of alignments at time. Filter-based alignment is also used to distinguish reads with perfect matches from those with one or more mismatches.

Still, filter-based alignment approaches have several problems. When new sequence libraries are introduced, the entire multistage alignment process must be repeated to update results. Next, information about reads that have multiple matches in different libraries, or perfect matches and non-perfect matches within a library are lost. Finally, because alignment formats between programs differ and good methods for organizing alignment data do not exist, it is hard to compare alignments between multiple samples. This last issue also creates challenges for linking alignments to the original sequence data and formatting information for other tools.

As previously noted, solving the above problems requires that alignment data be organized in ways that facilitate computation. HDF5 provides the foundation to organize and store both read and alignment data to enable different kinds of data comparisons. This ability is demonstrated by the following two examples.

In the first example (left), reads from different sequencing platforms (SOLiD, Illumina, 454) were stored in HDF5. Illumina RNA-Seq reads from three different samples were aligned to the human genome and annotations from a UCSC GFF (genome file format) file were applied to define gene boundaries. The example shows the alignment data organized into three HDF5 files, one per sample, but in reality the data could have been stored in a single file or files organized in other ways. One of HDF's strengths is that the HDF5 I/O library can query multiple files as if they were a single file, providing the ability to create the high-level data organizations that are the most appropriate for a particular application or use case. With reads and alignments structured in these files, it is a simple matter to integrate data to view base (color) compositions for reads from different sequencing platforms, compare alternative splicing between samples, and select a subset of alignments from a specific genomic region, or gene, in a "wig" format for viewing in a tool like the UCSC genome browser.

The second example (right) focuses on how organizing alignment data in HDF5 can change how differential alignment problems are approached. When data are organized according to a model that defines granularity and relationships, it becomes easier to compute all alignments between reads and multiple reference sources, than think about how to perform differential alignments and implement the process. In this case, a set of reads (obtained from cDNA) are aligned to primers, QC data (ribosomal RNA [rRNA] and mitochondrial DNA [mtDNA]), miRBase, refseq transcripts, the human genome, and a library of exon junctions. During alignment up to three mismatches are tolerated between a read and its hit. Alignment data are stored in HDF5 and, because the data were not filtered, a greater variety of questions can be asked. Subtractive questions mimic the differential pipeline where alignments are used to filter reads from subsequent steps. At the same time, we can also ask "biological" questions about the number of reads that came from rRNA or mtDNA or from genes in the genome or exon junctions. And for these questions, we can examine the match quality between each read and its matching sequence in the reference data sources, without having to reprocess the same data multiple times.

The above examples demonstrate the benefits of being able to organize data into structures that are amenable to computation. When data are properly structured, new approaches that expand the ways in which data are analyzed can be implemented. HDF5 and its library of software routines move the development process from activities associated with optimizing the low level infrastructures needed to support such systems to designing and testing different data models and exploiting their features.

The final post of this series will cover why we chose to work with HDF5 technology.