Davide Campagna, Alessandro Albiero, Alessandra Bilardi, Elisa Caniato, Claudio Forcato, Svetlin Manavski, Nicola Vitulo, Giorgio Valle.
Bioinformatics, Advance Access | doi:10.1093/bioinformatics/btp087 | PMID:19218350
Standard DNA alignment programs are inadequate to manage the data produced by new generation DNA sequencers. To answer this problem we developed PASS with the objective of improving execution time and sensitivity when compared with other available programs. PASS performs fast gapped and ungapped alignments of short DNA sequences onto a reference DNA, typically a genomic sequence. It is designed to handle a huge amount of reads such as those generated by Solexa, SOLiD or 454 technologies. The algorithm is based on a data structure that holds in RAM the index of the genomic positions of "seed" words (typically 11-12 bases) as well as an index of the precomputed scores of short words (typically 7-8 bases) aligned against each other. After building the genomic index, the program scans every query sequence performing 3 steps: 1) finds matching seed words in the genome; 2) for every match checks the precomputed alignment of the short flanking regions; 3) if passes step 2, then it performs an exact dynamic alignment of a narrow region around the match. The performance of the program is very striking both for sensitivity and speed. For instance, gap alignment is achieved hundreds of times faster than BLAST and several times faster than SOAP, especially when gaps are allowed. Furthermore, PASS has a higher sensitivity when compared with the other available programs.
U. Mirsaidov, W. Timp, X. Zou, V. Dimitrov, K. Schulten, A.P. Feinberg, G. Timp.
Biophysical Journal, 96, L32-L34, (2009) | doi:10.1016/j.bpj.2008.12.3760 | PMID:19217843
Methylation of cytosine is a covalent modification of DNA that can be used to silence genes, orchestrating a myriad of biological processes including cancer. We have discovered that a synthetic nanopore in a membrane comparable in thickness to a protein binding site can be used to detect methylation. We observe a voltage threshold for permeation of methylated DNA through a <2 nm diameter pore, which we attribute to the stretching transition; this can differ by >1 V/20 nm depending on the methylation level, but not the DNA sequence.
Gabriella Rozera, Isabella Abbate, Alessandro Bruselles, Crhysoula Vlassi, Gianpiero D'Offizi, Pasquale Narciso, Giovanni Chillemi, Mattia Prosperi, Giuseppe Ippolito, Maria R Capobianchi.
Retrovirology 6, 15 (2009) | doi:10.1186/1742-4690-6-15 | PMID:19216757
Background
Virus-associated cell membrane proteins acquired by HIV-1 during budding may give information on the cellular source of circulating virions. In the present study, by applying immunosorting of the virus and of the cells with antibodies targeting monocyte (CD36) and lymphocyte (CD26) markers, it was possible to directly compare HIV-1 quasispecies archived in circulating monocytes and T lymphocytes with that present in plasma virions originated from the same cell types. Five chronically HIV-1 infected patients who underwent therapy interruption after prolonged HAART were enrolled in the study. The analysis was performed by the powerful technology of ultra-deep pyrosequencing after PCR amplification of part of the env gene, coding for the viral glycoprotein (gp) 120, encompassing the tropism-related V3 loop region. V3 aminoacid sequences were used to establish heterogeneity parameters, to build phylogenetic trees and to predict co-receptor usage.
Results
The heterogeneity of proviral and viral genomes derived from monocytes was higher than that of T-lymphocyte origin. Both monocytes and T lymphocytes might contribute to virus rebounding in the circulation after therapy interruptions, but other virus sources might also be involved. In addition, both proviral and circulating viral sequences from monocytes and T lymphocytes were predictive of a predominant R5 coreceptor usage, but minor variants, segregating from the most frequent quasispecies variants, were present. In particular, in proviral genomes harboured by monocytes, minority variant clusters, with predicted X4 phenotype, were found.
Conclusions
This study provided the first direct comparison between the HIV-1 quasispecies archived as provirus in circulating monocytes and T lymphocytes with that of plasma virions replicating in the same cell types. Ultra-deep pyrosequencing generated data of some order of magnitude higher than any previously obtained with conventional approaches. Next generation sequencing allowed the analysis of previously inaccessible aspects of HIV-1 quasispecies, such as coreceptor usage of minority variants present in archived proviral sequences and in actually replicating virions, that may have clinical and therapeutic relevance
Ann C. Palmenberg, David Spiro, Ryan Kuzmickas, Shiliang Wang, Appolinaire Djikeng, Jennifer A. Rathe, Claire M. Fraser-Liggett, Stephen B. Liggett.
Science, Science Express | DOI:10.1126/science.1165557 | PMID:19213880
Infection by human rhinoviruses (HRVs) is a major cause of upper and lower respiratory tract disease worldwide and displays significant phenotypic variation. We examined diversity by completing the genome sequences for all known serotypes (n = 99). Superimposition of capsid crystal structure and optimal-energy RNA configurations established alignments and phylogeny. These revealed conserved motifs, clade-specific diversity including a potential new species (HRV-D), mutations in field isolates, and recombination. In analogy with poliovirus, a hypervariable 5'UTR tract may affect virulence. A configuration consistent with nonscanning internal ribosome entry was found in all HRVs and may account for rapid translation. The data density from complete sequences of the reference HRVs provided high resolution for this degree of modeling and serves as a platform for full genome-based epidemiologic studies and antiviral or vaccine development.
Axel Visel, Matthew J. Blow, Zirong Li, Tao Zhang, Jennifer A. Akiyama, Amy Holt, Ingrid Plajzer-Frick, Malak Shoukry, Crystal Wright, Feng Chen, Veena Afzal, Bing Ren, Edward M. Rubin, Len A. Pennacchio.
Nature 457, 854-858 (2009) | doi:10.1038/nature07730 | PMID:19212405
A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover because they are scattered among the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here we present the results of chromatin immunoprecipitation with the enhancer-associated protein p300 followed by massively parallel sequencing, and map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases demonstrated reproducible enhancer activity in the tissues that were predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities, and suggest that such data sets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.
