Improved assembly procedure of viral RNA genomes amplified with Phi29 polymerase from new generation sequencing data

BACKGROUND: New sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. However, the use of these new methods can require an amplification of viral RNA prior to the sequencing. Among all the available methods, the procedure based on the use of Phi29 polymerase produces a huge amount of amplified DNA. However, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. The pre-process method proposed in this study strongly limits the negative impact of chimeric reads in order to obtain the full-length of viral genomes. FINDINGS: Three different assembly softwares (ABySS, Ray and SPAdes) were tested for their ability to correctly assemble the full-length of viral genomes. Although in all cases, our pre-processed method improved genome assembly, only its combination with the use of SPAdes allowed us to obtain the full-length of the viral genomes tested in one contig. CONCLUSIONS: The proposed pipeline is able to overcome drawbacks due to the generation of chimeric reads during the amplification of viral RNA which considerably improves the assembling of full-length viral genomes.

Chromosome 19p13. 3 deletion in a child with Peutz-Jeghers syndrome, congenital heart defect, high myopia, learning difficulties and dysmorphic features: clinical and molecular characterization of a new contiguous gene syndrome

The Peutz-Jeghers syndrome (PJS) is an autosomal-dominant hamartomatous polyposis syndrome characterized by mucocutaneous pigmentation, gastrointestinal polyps and the increased risk of multiple cancers. The causative point mutation in the STK11 gene of most patients accounts for about 30 percent of the cases of partial and complete gene deletion. This is a report on a girl with PJS features, learning difficulties, dysmorphic features and cardiac malformation, bearing a de novo 1.1 Mb deletion at 19p13.3. This deletion encompasses at least 47 genes, including STK11. This is the first report on 19p13.3 deletion associated with a PJS phenotype, as well as other atypical manifestations, thereby implying a new contiguous gene syndrome.

The structure and function analysis of duplicate genes in Merlin strains of human cytomegalovirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To determine the genes in which exist overlapping ORF in Merlin strains of human cytomegalovirus, and to reveal their structure and functional characteristics.</p><p><b>METHODS</b>We search for overlapping genes of ORF in HCMV Merlin strains' whole genome by Bioinformatics methods, analyzing coding sequence CDS and starting and ending sites of ORF, calculating the length of CDS and ORF, analyzing the molecular weight of encoding protein, overlapping length and coding direction of protein, identifying overlapping sequences and overlapping types, analyzing the expression phase of overlapping genes and the function of proteins.</p><p><b>RESULTS</b>There were 39 overlapping ORF genes in HCMV Merlin strains, accounting for 23% of total genes. Among these 39 genes, there are 13 IE genes, 9 E genes and 17 L genes, which can be divided into 16 contigs. There are 11 contigs when two genes overlap, with 3 contigs in three genes overlapping, and 2 contigs in four genes overlapping. The functions of overlapping genes are widely.</p><p><b>CONCLUSION</b>We found that there are a lot of complex overlapping genes in HCMV Merlin strains, which are basis for further study of the transcription and translation mechanism of overlapping genes.</p>

Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol

In the finishing phase of the Chromobacterium violaceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were correctly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that the use of PACE, followed by combinatory PCR, accelerated the finishing phase of the C. violaceum genome project by at least 40 per cent