ALBERT

Description: Background Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here ‘nanopore sequencing’) has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. Results The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. Conclusion Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

Description: Background We have recently identified two large families of extinct transposable elements termed Short Interspersed DEgenerated Retroposons (SIDERs) in the parasitic protozoan Leishmania major. The characterization of SIDER elements was limited to the SIDER2 subfamily, although members of both subfamilies have been shown to play a role in the regulation of gene expression at the post-transcriptional level. Apparent functional domestication of SIDERs prompted further investigation of their characterization, dissemination and evolution throughout the Leishmania genus, with particular attention to the disregarded SIDER1 subfamily. Results Using optimized statistical profiles of both SIDER1 and SIDER2 subgroups, we report the first automated and highly sensitive annotation of SIDERs in the genomes of L. infantum, L. braziliensis and L. major. SIDER annotations were combined to in-silico mRNA extremity predictions to generate a detailed distribution map of the repeat family, hence uncovering an enrichment of antisense-oriented SIDER repeats between the polyadenylation and trans-splicing sites of intergenic regions, in contrast to the exclusive sense orientation of SIDER elements within 3'UTRs. Our data indicate that SIDER elements are quite uniformly dispersed throughout all three genomes and that their distribution is generally syntenic. However, only 47.4% of orthologous genes harbor a SIDER element in all three species. There is evidence for species-specific enrichment of SIDERs and for their preferential association, especially for SIDER2s, with different metabolic functions. Investigation of the sequence attributes and evolutionary relationship of SIDERs to other trypanosomatid retroposons reveals that SIDER1 is a truncated version of extinct autonomous ingi-like retroposons (DIREs), which were functional in the ancestral Leishmania genome. Conclusion A detailed characterization of the sequence traits for both SIDER subfamilies unveils major differences. The SIDER1 subfamily is more heterogeneous and shows an evolutionary link with vestigial DIRE retroposons as previously observed for the ingi/RIME and L1Tc/NARTc couples identified in the T. brucei and T. cruzi genomes, whereas no identified DIREs are related to SIDER2 sequences. Although SIDER1s and SIDER2s display equivalent genomic distribution globally, the varying degrees of sequence conservation, preferential genomic disposition, and differential association to orthologous genes allude to an intricate web of SIDER assimilation in these parasitic organisms.

Description: Background Leishmania parasites cause a diverse spectrum of diseases in humans ranging from spontaneously healing skin lesions (e.g., L. major) to life-threatening visceral diseases (e.g., L. infantum). The high conservation in gene content and genome organization between Leishmania major and Leishmania infantum contrasts their distinct pathophysiologies, suggesting that highly regulated hierarchical and temporal changes in gene expression may be involved. Results We used a multispecies DNA oligonucleotide microarray to compare whole-genome expression patterns of promastigote (sandfly vector) and amastigote (mammalian macrophages) developmental stages between L. major and L. infantum. Seven per cent of the total L. infantum genome and 9.3% of the L. major genome were differentially expressed at the RNA level throughout development. The main variations were found in genes involved in metabolism, cellular organization and biogenesis, transport and genes encoding unknown function. Remarkably, this comparative global interspecies analysis demonstrated that only 10–12% of the differentially expressed genes were common to L. major and L. infantum. Differentially expressed genes are randomly distributed across chromosomes further supporting a posttranscriptional control, which is likely to involve a variety of 3'UTR elements. Conclusion This study highlighted substantial differences in gene expression patterns between L. major and L. infantum. These important species-specific differences in stage-regulated gene expression may contribute to the disease tropism that distinguishes L. major from L. infantum.

Description: Background Leishmania and other members of the Trypanosomatidae family diverged early on in eukaryotic evolution and consequently display unique cellular properties. Their apparent lack of transcriptional regulation is compensated by complex post-transcriptional control mechanisms, including the processing of polycistronic transcripts by means of coupled trans-splicing and polyadenylation. Trans-splicing signals are often U-rich polypyrimidine (poly(Y)) tracts, which precede AG splice acceptor sites. However, as opposed to higher eukaryotes there is no consensus polyadenylation signal in trypanosomatid mRNAs. Results We refined a previously reported method to target 5' splice junctions by incorporating the pyrimidine content of query sequences into a scoring function. We also investigated a novel approach for predicting polyadenylation (poly(A)) sites in-silico, by comparing query sequences to polyadenylated expressed sequence tags (ESTs) using position-specific scanning matrices (PSSMs). An additional analysis of the distribution of putative splice junction to poly(A) distances helped to increase prediction rates by limiting the scanning range. These methods were able to simplify splice junction prediction without loss of precision and to increase polyadenylation site prediction from 22% to 47% within 100 nucleotides. Conclusion We propose a simplified trans-splicing prediction tool and a novel poly(A) prediction tool based on comparative sequence analysis. We discuss the impact of certain regions surrounding the poly(A) sites on prediction rates and contemplate correlating biological mechanisms. This work aims to sharpen the identification of potentially functional untranslated regions (UTRs) in a large-scale, comparative genomics framework.