Noah Fahlgren, Christopher M. Sullivan, Kristin D. Kasschau, Elisabeth J. Chapman, Jason S. Cumbie, Taiowa A. Montgomery, Sunny D. Gilbert, Mark Dasenko, Tyler W.H. Backman, Scott A. Givan, James C. Carrington.
RNA 15 992-1002 (2009) | doi:10.1261/rna.1473809 | PMID:19307293
The advent of high-throughput sequencing (HTS) methods has enabled direct approaches to quantitatively profile small RNA populations. However, these methods have been limited by several factors, including representational artifacts and lack of established statistical methods of analysis. Furthermore, massive HTS data sets present new problems related to data processing and mapping to a reference genome. Here, we show that cluster-based sequencing-by-synthesis technology is highly reproducible as a quantitative profiling tool for several classes of small RNA from Arabidopsis thaliana. We introduce the use of synthetic RNA oligoribonucleotide standards to facilitate objective normalization between HTS data sets, and adapt microarray-type methods for statistical analysis of multiple samples. These methods were tested successfully using mutants with small RNA biogenesis (miRNA-defective dcl1 mutant and siRNA-defective dcl2 dcl3 dcl4 triple mutant) or effector protein (ago1 mutant) deficiencies. Computational methods were also developed to rapidly and accurately parse, quantify, and map small RNA data.
Tianjiao Chu, Kimberly Bunce, W. Allen Hogge, David G. Peters.
Bioinformatics, Advance Access | doi:10.1093/bioinformatics/btp156 | PMID:19307238
There is currently great interest in the development of methods for the minimally invasive diagnosis of fetal genetic disease using cell-free DNA from maternal plasma samples obtained in the first trimester of pregnancy. With the rapid development of high-throughput sequencing technology, the possibility of detecting the presence of trisomy fetal genomes in the maternal plasma DNA sample has recently been explored (Fan, et al., 2008). The major concern of this whole genome sequencing approach is that, while detecting the karyotype of the fetal genome from the maternal plasma requires extremely high accuracy of copy number estimation, the majority of available high throughput sequencing technologies require PCR and are subject to the substantial bias that is inherent to the PCR process. We introduce a novel and sophisticated statistical model for the whole genome sequencing data, and based on this model, develop a highly sensitive method of Minimally Invasive Karyotyping (MINK) for the Diagnosis of Fetal Genetic Disease. Specifically we demonstrate, by applying our statistical method to ultra high-throughput whole sequencing data, that trisomy 21 can be detected in a minor ("fetal") genome when it is mixed into a major ("maternal") background genome at frequencies as low as 5%. This observation provides additional proof of concept and justification for the further development of this method towards its eventual clinical application. Here we describe the statistical and experimental methods that illustrate this approach and discuss future directions for technical development and potential clinical applications.
Piotr A. Ziolkowski, Grzegorz Koczyk, Lukasz Galganski, Jan Sadowski.
Nucleic Acids Research, Advance Access | doi:10.1093/nar/gkp183 | PMID:19305000
Large differences in plant genome sizes are mainly due to numerous events of insertions or deletions (indels). The balance between these events determines the evolutionary direction of genome changes. To address the question of what phenomena trigger these alterations, we compared the genomic sequences of two Arabidopsis thaliana lines, Columbia (Col) and Landsberg erecta (Ler). Based on the resulting alignments large indels (>100 bp) within these two genomes were analysed. There are ~8500 large indels accounting for the differences between the two genomes. The genetic basis of their origin was distinguished as three main categories: unequal recombination (Urec)-derived, illegitimate recombination (Illrec)-derived and transposable elements (TE)-derived. A detailed study of their distribution and size variation along chromosomes, together with a correlation analyses, allowed us to demonstrate the impact of particular recombination-based mechanisms on the plant genome evolution. The results show that unequal recombination is not efficient in the removal of TEs within the pericentromeric regions. Moreover, we discovered an unexpectedly high influence of large indels on gene evolution pointing out significant differences between the various gene families. For the first time, we present convincing evidence that somatic events do play an important role in plant genome evolution.
Karla D. Passalacqua, Anjana Varadarajan, Brian D. Ondov, David T. Okou, Michael E. Zwick, Nicholas H. Bergman.
J. Bacteriol., JB Accepts | doi:10.1128/JB.00122-09 | PMID:19304856
Although gene expression has been studied in bacteria for decades, many aspects of the bacterial transcriptome remain poorly understood. Transcript structure, operon linkages, and absolute abundance information all provide valuable insights into gene function and regulation, but none has ever been determined on a genome-wide scale for any bacterium. Indeed, these aspects of the prokaryotic transcriptome have only been explored on a large scale in a few instances, and consequently little is known about the absolute composition of the mRNA population within a bacterial cell. Here we report the use of a high-throughput sequencing-based approach (RNA-Seq) in assembling the first comprehensive, single-nucleotide resolution view of a bacterial transcriptome. We sampled the Bacillus anthracis transcriptome under a variety of growth conditions, and showed that these data provide an accurate and high-resolution map of transcript start sites and operon structure throughout the genome. Further, the sequence data identified previously unannotated regions with significant transcriptional activity, and enhanced the accuracy of existing genome annotations. Finally, our data provide estimates of absolute transcript abundance, and suggest there is significant transcriptional heterogeneity within a clonal, synchronized bacterial population. Overall, our results offer an unprecedented view of gene expression and regulation in a bacterial cell.
Julia Sandberg, Patrik L. Ståhl, Afshin Ahmadian, Magnus K. Bjursell Joakim Lundeberg.
Nucleic Acids Research, Advance Access | doi:10.1093/nar/gkp188 | PMID:19304748
Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.
