Computational protocol: A novel species of torque teno mini virus (TTMV) in gingival tissue from chronic periodontitis patients

Similar protocols

Protocol publication

[…] The samples were vortexed with small magnetic beads for 5 min and freeze-thawed three times. After centrifugation (10 min at 13,000 × g), 300 μl of supernatant was collected and filtered through a 0.45-μm filter to remove eukaryotic and bacterial cell-sized particles. To digest unprotected nucleic acids (not in viral capsids), a mixture of DNases (Turbo DNase from Ambion, MA, USA, Baseline-ZERO from Epicentre, IL, USA, and benzonase from Novagen, Darmstadt, Germany) and RNase (Thermo Fisher Scientific, MA, USA) was added to the filtrates, which were enriched in viral particles, and the samples were incubated at 37 °C for 90 min. Viral nucleic acids were then extracted using a QIAamp viral RNA extraction kit (QIAGEN, Dusseldorf, Germany) according to the manufacturer’s instructions, protected from degradation by the addition of an RNase inhibitor (Thermo Fisher Scientific, MA, USA), and stored at −80 °C for future processing. Viral nucleic acid libraries containing both DNA and RNA viral sequences were constructed by random RT-PCR amplification based on the Nextera XT DNA Sample Preparation Kit (Illumina, CA, USA). The libraries were sequenced using the MiSeq platform. The sequencing reads were assigned to 48 data bin-based barcodes. Trimmed sequences from each group were assembled into contigs using the method described by Eric in 2011, with a criterion at least 95% identity over 35-bp to merge two fragments. The assembled contigs and singlet sequences were then compared to GenBank using BLASTx. Sequences with E values of ≤10−5 in a BLASTx search were classified as likely originating from a eukaryotic virus, bacterium, phage, eukaryote, other, or unknown based on the taxonomic origin of the sequence with the best E value. [...] The genome sequence of the newly discovered human anellovirus isolated in this study was aligned using ClustalW with the default settings, and the aligned sequences were trimmed to match the human anellovirus sequences obtained from the human anelloviruses detected using ClustalW. Sequence analyses were performed with MegAlign software (DNAStar Inc., Madison, WI, USA). A phylogenetic tree was constructed using the neighbor-joining method with nucleotide p distances and 1,000 bootstrap replicates in the Molecular Evolutionary Genetics Analysis program (MEGA, version 4.0, USA) and by inputting the alignment of the ORF1 genome sequences. Bootstrap values are indicated at each branching point. A % similarity analysis was also conducted using the MEGA program. […]

Pipeline specifications

Software tools BLASTX, Clustal W, MEGA
Applications Phylogenetics, Metagenomic sequencing analysis
Organisms TTV-like mini virus, Homo sapiens, Human poliovirus 1 Mahoney
Diseases Periodontitis, Chronic Periodontitis
Chemicals Nucleotides