Showing papers by "Sam Griffiths-Jones published in 2012"

Reducing ligation bias of small RNAs in libraries for next generation sequencing

Abstract: Background The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing.

Evolution and function of the extended miR-2 microRNA family.

Abstract: MicroRNAs are essential post-transcriptional regulators. Many animal microRNAs are clustered in the genome, and it has been shown that clustered microRNAs may be transcribed as a single transcript. Polycistronic microRNAs are often members of the same family, suggesting a role of tandem duplication in the emergence of clusters. The mir-2 microRNA family is the largest in Drosophila melanogaster, with 8 members that are mostly clustered in the genome. Previous studies suggest that the copy number and genomic distribution of mir-2 family members has been subject to significant change during evolution. The effects of such changes on their function are still unknown. Here we study the evolution of function in the mir-2 family. Our analyses show that, in spite of the change in number and organization among invertebrates, most mir-2 loci produce very similar mature microRNA products. Multiple mature miR-2 sequences are predicted to target genes involved in neural development in Drosophila. These targeting properties are conserved in the distant species Caenorhabditis elegans. Duplication followed by functional diversification is frequent during protein-coding gene evolution. However, our results suggest that the production of microRNA clusters by gene duplication rarely involves functional changes. This pattern of functional redundancy among clustered paralogous microRNAs reflects birth-and-death evolutionary dynamics. However, we identified a small number of mir-2 sequences in Drosophila that may have undergone functional shifts associated with genomic rearrangements. Therefore, redundancy in microRNA families may facilitate the acquisition of novel functional features.

MicroRNAs from the same precursor have different targeting properties

Abstract: Background The processing of a microRNA results in an intermediate duplex of two potential mature products that derive from the two arms (5′ and 3′) of the precursor hairpin. It is often suggested that one of the sequences is degraded and the other is incorporated into the RNA-induced silencing complex. However, both precursor arms may give rise to functional levels of mature microRNA and the dominant product may change from species to species, from tissue to tissue, or between developmental stages. Therefore, both arms of the precursor have the potential to produce functional mature microRNAs.

Mitochondrial pseudogenes in the nuclear genomes of Drosophila.

Abstract: Mitochondrial pseudogenes in nuclear chromosomes (numts) have been detected in the genomes of a diverse range of eukaryotic species. However, the numt content of different genomes and their properties is not uniform, and study of these differences provides insight into the mechanisms and dynamics of genome evolution in different organisms. In the genus Drosophila, numts have previously only been identified on a genome-wide scale in the melanogaster subgroup. The present study extends the identification to 11 species of the Drosophila genus. We identify a total of 302 numts and show that the numt complement is highly variable in Drosophilids, ranging from just 4 in D. melanogaster to 67 in D. willistoni, broadly correlating with genome size. Many numts have undergone large-scale rearrangements in the nucleus, including interruptions, inversions, deletions and duplications of sequence of variable size. Estimating the age of the numts in the nucleus by phylogenetic tree reconstruction reveals the vast majority of numts to be recent gains, 90% having arisen on terminal branches of the species tree. By identifying paralogs and counting duplications among the extant numts we estimate that 23% of extant numts arose through post-insertion duplications. We estimate genus average rates of insertion of 0.75 per million years, and a duplication rate of 0.010 duplications per numt per million years.

Detection of microRNAs in color space

Abstract: Motivation: Deep sequencing provides inexpensive opportunities to characterize the transcriptional diversity of known genomes. The AB SOLiD technology generates millions of short sequencing reads in color-space; that is, the raw data is a sequence of colors, where each color represents 2 nt and each nucleotide is represented by two consecutive colors. This strategy is purported to have several advantages, including increased ability to distinguish sequencing errors from polymorphisms. Several programs have been developed to map short reads to genomes in color space. However, a number of previously unexplored technical issues arise when using SOLiD technology to characterize microRNAs. Results: Here we explore these technical difficulties. First, since the sequenced reads are longer than the biological sequences, every read is expected to contain linker fragments. The color-calling error rate increases toward the 3′ end of the read such that recognizing the linker sequence for removal becomes problematic. Second, mapping in color space may lead to the loss of the first nucleotide of each read. We propose a sequential trimming and mapping approach to map small RNAs. Using our strategy, we reanalyze three published insect small RNA deep sequencing datasets and characterize 22 new microRNAs. Availability and implementation: A bash shell script to perform the sequential trimming and mapping procedure, called SeqTrimMap, is available at: http://www.mirbase.org/tools/seqtrimmap/ Contact: antonio.marco@manchester.ac.uk Supplementary information:Supplementary data are available at Bioinformatics online.