Ancient DNA studies: new perspectives on old samples

In spite of past controversies, the field of ancient DNA is now a reliable research area due to recent methodological improvements. A series of recent large-scale studies have revealed the true potential of ancient DNA samples to study the processes of evolution and to test models and assumptions commonly used to reconstruct patterns of evolution and to analyze population genetics and palaeoecological changes. Recent advances in DNA technologies, such as next-generation sequencing make it possible to recover DNA information from archaeological and paleontological remains allowing us to go back in time and study the genetic relationships between extinct organisms and their contemporary relatives. With the next-generation sequencing methodologies, DNA sequences can be retrieved even from samples (for example human remains) for which the technical pitfalls of classical methodologies required stringent criteria to guaranty the reliability of the results. In this paper, we review the methodologies applied to ancient DNA analysis and the perspectives that next-generation sequencing applications provide in this field

Non-random retention of protein-coding overlapping genes in Metazoa

<p>Abstract</p> <p>Background</p> <p>Although the overlap of transcriptional units occurs frequently in eukaryotic genomes, its evolutionary and biological significance remains largely unclear. Here we report a comparative analysis of overlaps between genes coding for well-annotated proteins in five metazoan genomes (human, mouse, zebrafish, fruit fly and worm).</p> <p>Results</p> <p>For all analyzed species the observed number of overlapping genes is always lower than expected assuming functional neutrality, suggesting that gene overlap is negatively selected. The comparison to the random distribution also shows that retained overlaps do not exhibit random features: antiparallel overlaps are significantly enriched, while overlaps lying on the same strand and those involving coding sequences are highly underrepresented. We confirm that overlap is mostly species-specific and provide evidence that it frequently originates through the acquisition of terminal, non-coding exons. Finally, we show that overlapping genes tend to be significantly co-expressed in a breast cancer cDNA library obtained by 454 deep sequencing, and that different overlap types display different patterns of reciprocal expression.</p> <p>Conclusion</p> <p>Our data suggest that overlap between protein-coding genes is selected against in Metazoa. However, when retained it may be used as a species-specific mechanism for the reciprocal regulation of neighboring genes. The tendency of overlaps to involve non-coding regions of the genes leads to the speculation that the advantages achieved by an overlapping arrangement may be optimized by evolving regulatory non-coding transcripts.</p

The germline of the malaria mosquito produces abundant miRNAs, endo-siRNAs, piRNAs and 29-nt small RNA

BACKGROUND Small RNAs include different classes essential for endogenous gene regulation and cellular defence against genomic parasites. However, a comprehensive analysis of the small RNA pathways in the germline of the mosquito Anopheles gambiae has never been performed despite their potential relevance to reproductive capacity in this malaria vector. RESULTS We performed small RNA deep sequencing during larval and adult gonadogenesis and find that they predominantly express four classes of regulatory small RNAs. We identified 45 novel miRNA precursors some of which were sex-biased and gonad-enriched , nearly doubling the number of previously known miRNA loci. We also determine multiple genomic clusters of 24-30 nt Piwi-interacting RNAs (piRNAs) that map to transposable elements (TEs) and 3'UTR of protein coding genes. Unusually, many TEs and the 3'UTR of some endogenous genes produce an abundant peak of 29-nt small RNAs with piRNA-like characteristics. Moreover, both sense and antisense piRNAs from TEs in both Anopheles gambiae and Drosophila melanogaster reveal novel features of piRNA sequence bias. We also discovered endogenous small interfering RNAs (endo-siRNAs) that map to overlapping transcripts and TEs. CONCLUSIONS This is the first description of the germline miRNome in a mosquito species and should prove a valuable resource for understanding gene regulation that underlies gametogenesis and reproductive capacity. We also provide the first evidence of a piRNA pathway that is active against transposons in the germline and our findings suggest novel piRNA sequence bias. The contribution of small RNA pathways to germline TE regulation and genome defence in general is an important finding for approaches aimed at manipulating mosquito populations through the use of selfish genetic elements

Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays

The Saccharopolyspora erythraea genome sequence, recently published, presents considerable divergence from those of streptomycetes in gene organization and function, confirming the remarkable potential of S. erythraea for producing many other secondary metabolites in addition to erythromycin. In order to investigate, at whole transcriptome level, how S. erythraea genes are modulated, a DNA microarray was specifically designed and constructed on the S. erythraea strain NRRL 2338 genome sequence, and the expression profiles of 6494 ORFs were monitored during growth in complex liquid medium

Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

AbstractThe early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration

Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer

<p>Abstract</p> <p>Background</p> <p>A new priority in genome research is large-scale resequencing of genes to understand the molecular basis of hereditary disease and cancer. We assessed the ability of massively parallel pyrosequencing to identify sequence variants in pools. From a large collection of human PCR samples we selected 343 PCR products belonging to 16 disease genes and including a large spectrum of sequence variations previously identified by Sanger sequencing. The sequence variants included SNPs and small deletions and insertions (up to 44 bp), in homozygous or heterozygous state.</p> <p>Results</p> <p>The DNA was combined in 4 pools containing from 27 to 164 amplicons and from 8,9 to 50,8 Kb to sequence for a total of 110 Kb. Pyrosequencing generated over 80 million base pairs of data. Blind searching for sequence variations with a specifically designed bioinformatics procedure identified 465 putative sequence variants, including 412 true variants, 53 false positives (in or adjacent to homopolymeric tracts), no false negatives. All known variants in positions covered with at least 30× depth were correctly recognized.</p> <p>Conclusion</p> <p>Massively parallel pyrosequencing may be used to simplify and speed the search for DNA variations in PCR products. Our results encourage further studies to evaluate molecular diagnostics applications.</p

Complete genome sequence of a serotype 11A, ST62 Streptococcus pneumoniae invasive isolate

<p>Abstract</p> <p>Background</p> <p><it>Streptococcus pneumoniae </it>is an important human pathogen representing a major cause of morbidity and mortality worldwide. We sequenced the genome of a serotype 11A, ST62 <it>S. pneumoniae </it>invasive isolate (AP200), that was erythromycin-resistant due to the presence of the <it>erm</it>(TR) determinant, and carried out analysis of the genome organization and comparison with other pneumococcal genomes.</p> <p>Results</p> <p>The genome sequence of <it>S. pneumoniae </it>AP200 is 2,130,580 base pair in length. The genome carries 2216 coding sequences (CDS), 56 tRNA, and 12 rRNA genes. Of the CDSs, 72.9% have a predicted biological known function. AP200 contains the pilus islet 2 and, although its phenotype corresponds to serotype 11A, it contains an 11D capsular locus. Chromosomal rearrangements resulting from a large inversion across the replication axis, and horizontal gene transfer events were observed. The chromosomal inversion is likely implicated in the rebalance of the chromosomal architecture affected by the insertions of two large exogenous elements, the <it>erm</it>(TR)-carrying Tn<it>1806 </it>and a functional prophage designated ϕSpn_200. Tn<it>1806 </it>is 52,457 bp in size and comprises 49 ORFs. Comparative analysis of Tn<it>1806 </it>revealed the presence of a similar genetic element or part of it in related species such as <it>Streptococcus pyogenes </it>and also in the anaerobic species <it>Finegoldia magna, Anaerococcus prevotii </it>and <it>Clostridium difficile</it>. The genome of ϕSpn_200 is 35,989 bp in size and is organized in 47 ORFs grouped into five functional modules. Prophages similar to ϕSpn_200 were found in pneumococci and in other streptococcal species, showing a high degree of exchange of functional modules. ϕSpn_200 viral particles have morphologic characteristics typical of the <it>Siphoviridae </it>family and are capable of infecting a pneumococcal recipient strain.</p> <p>Conclusions</p> <p>The sequence of <it>S. pneumoniae </it>AP200 chromosome revealed a dynamic genome, characterized by chromosomal rearrangements and horizontal gene transfers. The overall diversity of AP200 is driven mainly by the presence of the exogenous elements Tn<it>1806 </it>and ϕSpn_200 that show large gene exchanges with other genetic elements of different bacterial species. These genetic elements likely provide AP200 with additional genes, such as those conferring antibiotic-resistance, promoting its adaptation to the environment.</p

A transcriptional sketch of a primary human breast cancer by 454 deep sequencing

Background: The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts. Results: We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas. Conclusion: Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling