PMC:7151644 / 6841-9042
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7151644","sourcedb":"PMC","sourceid":"7151644","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7151644","text":"2.2 RNA library construction, sequencing, and data analysis\nTotal RNA was extracted from blood, organ tissue, and fecal samples, as well as ticks, using Nucleo Spin RNA Blood (MN, Düren, Germany), RNeasy Plus Mini Kit (Qiagen, Valencia, California USA), RNeasy Plus Universal Mini Kit (Qiagen) and TRIzol LS Reagent (Invitrogen, Carlsbad, California, USA), respectively, following the manufacturer’s instructions. DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen). For RNA library construction, aliquots of RNA solution were pooled in equal quantity (Supplementary Table S2). The SMARTer Stranded Total RNA-Seq Kit v2 (TaKaRa, Dalian, China) and KAPA RNA HyperPrep Kit with RiboErase (HMR; KAPA, Wilmington, Massachusetts, USA) was used to construct RNA libraries from blood, organ tissue, and fecal samples, respectively. Ribosomal (r) RNA was removed using the Ribo-Zero-Gold (HMR) Kit (Illumina, San Diego, California, USA). Paired-end (150 bp) sequencing of each RNA library was performed on the HiSeqX10 platform (Illumina).\nBioinformatic analyses of the sequencing reads were undertaken as described previously (Shi et al. 2016a). In brief, adaptor- and quality-filtered sequencing reads were assembled de novo using the Trinity program (version 2.5.1). Viral contigs were identified by comparison (using blastn and Diamond blastx) to the NCBI non-redundant nucleotide (nt) and protein (nr) database with e-values set to 1 × 10−10 and 1 × 10−4, respectively. Likely contaminating viral sequences were excluded from the meta-transcriptomic data (Supplementary Table S2) using methods described previously (Asplund et al. 2019). In addition, the high frequency of retrovirus sequences was also excluded, as the majority of them probably were host genes, and some of them might be contaminating viral sequences, such as alpharetroviral and gammaretroviral sequences probably linked to laboratory components (Asplund et al. 2019). Finally, the putative viruses present in the blood, liver, spleen, lung, and kidney samples were confirmed by PCR. The quantity of the transcripts mapped to each viral contig was determined using the RSEM program (Li et al. 2010) implemented in Trinity.","divisions":[{"label":"title","span":{"begin":0,"end":59}},{"label":"p","span":{"begin":60,"end":1044}}],"tracks":[{"project":"2_test","denotations":[{"id":"32296543-31059795-144433899","span":{"begin":1641,"end":1645},"obj":"31059795"},{"id":"32296543-31059795-144433900","span":{"begin":1941,"end":1945},"obj":"31059795"},{"id":"32296543-20022975-144433901","span":{"begin":2172,"end":2176},"obj":"20022975"}],"attributes":[{"subj":"32296543-31059795-144433899","pred":"source","obj":"2_test"},{"subj":"32296543-31059795-144433900","pred":"source","obj":"2_test"},{"subj":"32296543-20022975-144433901","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#e693ec","default":true}]}]}}