Wednesday, January 28, 2009

The Next Generation Dilemma: Large Scale Data Analysis

Next week is the AGBT genome conference in Marco Island, Florida. At the conference we will present a poster on work we have been doing with Next Gen Sequencing data analysis. In this post we present the abstract. We'll post the poster when we return from sunny Florida.


The volumes of data that can be obtained from Next Generation DNA sequencing instruments make several new kinds of experiments possible and new questions amenable to study. The scale of subsequent analyses, however, presents a new kind of challenge. How do we get from a collection of several million short sequences of bases to genome-scale results? This process involves three stages of analysis that can be described as primary, secondary, and tertiary data analyses. At the first stage, primary data analysis, image data are converted to sequence data. In the middle stage, secondary data analysis, sequences are aligned to reference data to create application-specific data sets for each sample. In the final stage, tertiary data analysis, the data sets are compared to create experiment-specific results. Currently, the software for the primary analyses is provided by the instrument manufacturers and handled within the instrument itself, and when it comes to the tertiary analyses, many good tools already exist. However, between the primary and tertiary analyses lies a gap.

In RNA-Seq, the process of determining relative gene expression means that sequence data from multiple samples must go through the entire process of primary, secondary, and tertiary analysis. To do this work, researchers must puzzle through a diverse collection of early version algorithms that are combined into complicated workflows with steps producing complicated file formats. Command line tools such as MAQ, SOAP, MapReads, and BWA, have specialized requirements for formatted input and output and leave researchers with large data files that still require additional processing and formatting for tertiary analyses. Moreover, once reads are aligned, datasets need to be visualized and further refined for additional comparative analysis. We present a solution to these challenges that closes the gaps between primary, secondary, and tertiary analysis by showing results from a complete workflow system that includes data collection, processing and analysis for RNA-seq.

And, if you cannot be in sunny Florida, join us in Memphis where we will help kick off the ABRF conference with a workshop on Next Generation DNA Sequencing. I'm kicking the workshop off with a talk entitled "From Reads to Data Sets, Why Next Gen is Not Like Sanger Sequencing."

Wednesday, January 21, 2009

The Experts Agree

It depends what you are trying to do. That is the take home message in Genome Technology’s (GT) trouble-shooting guide on picking assembly and alignment algorithms for Next-Gen sequence data.

In the guide, the GT team asked nine Next-Gen sequencing and bioinformatics experts to answer six questions:
  1. How do you choose which alignment algorithm to use?
  2. How do you optimize your alignment algorithm for both high speed and low error rate?
  3. What approach do you use to handle mismatches or alignment gaps?
  4. How do you choose which assembly algorithm to use?
  5. Do you use mate-paired reads for de novo assembly? how?
  6. What impact does the quality of raw read data have on alignment or assembly? how do your algorithms enhance this?
Even a quick look at the questions shows us that many factors need to be considered in setting up a Next-Gen sequencing lab. Questions 1 and 4 point out that aligning sequences is different from assembling them. Other questions address issues related to the size of the data sets being compared, the quality of the data being analyzed, the kinds of information that can be obtained, and the computational approaches being used for different problems.

What the experts said

First, they all agree that different problems require different approaches and have different requirements. In the first question about which aligner to use, the most common response was “for what application and which instrument?” Fundamentally, SOLiD data are different from Illumina GA data which are different from 454 data. While the end results may all be sequences of A's, G's, C's, and T's; the data are derived in different ways because of the platform-specific twists in collecting the data (recall “Color Space, Flow Space, Sequence Space, or Outer Space). Not only are there platform-specific methods for interpreting raw data, multiple programs have been developed for each instrument with their own strengths and weaknesses in terms of speed, sensitivity, the kinds of data they use (color, base, or flow spaces, quality values, and paired end data), and the information that is finally produced. Hence, in addition to choosing a sequencing platform you also have to think about the sequencing application, or the kind of experiment, that will be performed. In gene expression studies, for example, an RNA-Seq experiment has different requirements in terms of aligning the data and interpreting the output than an experiment with Tag Profiling.

Overall the trouble-shooting guide discussed 17 total algorithms, eight for alignment, and nine for assembly (two of which were for Sanger methods). Even this selection wasn't a comprehensive list. When other sites [1, 2] and articles [3] are included and proprietary methods are factored in, over 20 algorithms are available. So what to do? Which is best?

That depends

Yes, the choice of algorithm ultimately depends on what you are trying to do. While we can agree that there is no best solution, we also know that is not a helpful response. What is needed is a way to test the suitability of different algorithms for different kinds of experiments and to represent data in standard ways so that the features of specific algorithms can be evaluated. Also, as this is a new field, standard requirements for how data should be aligned, defining a correct alignment, and what kinds of information are the most informative in describing alignments are still emerging. Some of the early programs are helping to define these requirements.

One program we've used, at Geopsiza, for identifying requirements is MAQ, a program for sequence alignment. As noted in previous blogs [MAQ attack], MAQ is a great general purpose tool. It provides comprehensive information about the data being aligned and details about alignments. MAQ works well for many applications including RNA-Seq, Tag Profiling, ChIP-Seq, and resequencing assays focused on SNP discovery. In performance tests, MAQ is slower than some of the newer programs, one of which is being developed by MAQ’s author, but MAQ is a good model for getting the right kinds of information, formatted in a decent way. Indeed MAQ was the most cited program in the GT guide.

Let’s return to the bigger issue. That is, how can we easily compare between algorithms? For that we need a system where one can easily define a standardized dataset and reference sequence, and have a platform where a new algorithm can be added and run from a common interface. Standard reports that present features of the alignments could then be used to compare programs and parameters.

The laboratory edition of GeneSifter supports these kinds of comparisons. The distributed system architecture allows one to quickly develop control scripts to run programs and format their output in figures and tables that make comparisons possible. With this kind of system in place, the challenges move from which program to run and how to run it, to how to get the right kinds of information and best display the data. To address these issues, Geospiza’s research and development team is working on projects focused on using technologies like HDF5 to create scalable standardized data models for storing information from alignment and assembly programs. Ultimately this work will make it easy to optimize Next-Gen sequencing applications and assays and compare between assorted programs.

3. Shendure J., Ji H., 2008. Next-generation DNA sequencing. Nat Biotechnol 26, 1135-1145.

Tuesday, January 6, 2009

From Reads to Data Sets, Why Next Gen is not like Sanger Sequencing

Time is running out! Be sure to register for the ABRF 2009 Next Generation DNA Sequencing Workshop to be held in Memphis TN Sat. Feb. 7, 2009. The day will include discussions from core lab directors and others about how to implement new sequencing technologies in a core lab environment. We'll consider how next generation sequencing differs from Sanger sequencing and devote much of the day learning about the practical impact of the differences.

An introduction:

Initially, DNA sequencing was performed to learn about sequences and structure of cloned genes. The first widely-used sequencing systems were based on the “Sanger” method. DNA was synthesized in the presence of chain terminating radioactive dideoxy-nucleotides (1). Mixtures of DNA fragments were separated by size using gel electrophoresis and the bases were identified and entered into a computer through manual techniques. Automated DNA sequencing instruments arrived later. These instruments made DNA sequencing thousands of times more efficient by detecting fluorescently labeled fragments and sending the information directly to a computer (2). For the first time, it became possible to sequence entire human genomes (3,4).

While highly successful, Sanger sequencing is cost prohibitive when it comes to deeper investigations of biological systems. Just as the questions investigated by Sanger sequencing shifted from single genes to entire genomes, the questions being asked by Next Generation techniques are changing as well. Questions related to transcription, for example, and promoter occupancy, can now be answered by using a massively parallel format to sample large collections of individual molecules. In effect, every RNA or DNA molecule might be sampled and counted. Not only are we looking at a “Next Generation” of DNA sequencing, we are looking a next generation of experimental techniques that answer different kinds of questions than those we asked before. These new technologies require fundamental changes in terms of experiment design and data analysis systems.

1) Sanger F., Nicklen S., Coulson A.R., 1977. “DNA sequencing with chain-terminating inhibitors.” Proc Natl Acad Sci U S A 74, 5463-5467.

2) Smith L.M., Sanders J.Z., Kaiser R.J., Hughes P., Dodd C., Connell C.R., Heiner C., Kent S.B., Hood L.E., 1986. “Fluorescence detection in automated DNA sequence analysis.” Nature 321, 674-679.

3) International Human Genome Sequencing Consortium, 2001. “Initial sequencing and analysis of the human genome.” Nature 409, 860-921.

4) Venter J.C., Adams M.D., Myers E.W., et. al. 2001. “The sequence of the human genome.” Science 291, 1304-1351.