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Pipeline publication

[…] b'nally, the pellet was resuspended in 250 \xce\xbcL RNase/DNase and protease-free water (Ambion), and viral RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) by using the manufacturer\xe2\x80\x99s protocols., Viral RNA (\xe2\x89\x880.9 \xce\xbcg) was fragmented by incubation at 94\xc2\xb0C for 8 min in 19.5 \xce\xbcL of Illumina fragmentation buffer 15016648 (Illumina, San Diego, CA, USA). A sequencing library was prepared from the sample RNA by using an Illumina TruSeq RNA Sample Preparation Kit v2 using the manufacturer\xe2\x80\x99s protocol (Illumina). The sample was sequenced on a HiSeq 1000 by using the 2 \xc3\x97 50 paired-end protocol. Reads in fastq format were quality filtered, and any adaptor sequences were removed by using Trimmomatic software (). The de novo assembly program ABySS () was used to assemble the reads into contigs, using several different sets of reads and k values from 20 to 40. In all samples, host reads were filtered out before de novo assembly. The longest contigs were selected, and reads were mapped back to the contigs by using bowtie2 () and visualized with the Integrated Genomics Viewer () to verify that the assembled contigs were correct. Total reads ranged from 1.5 to 12 million; the percentage of reads mapping to the virus genome in each sample ranged from 12% to 33%. (Details are available upon request from the authors.) , We deposited the complete genome sequences of Itaya virus and other group C viruses obtained for this study in GenBank under accession numbers KM092512-KM092514 and KM280924-KM280938. We used the neighbor-joining method available in MEGA5 () for phylogenetic analysis. The support for each node was determined by using 1,000 bootstrap replicates., We used established methods to obtain hyperimmune ascitic fluid for classic cross-neutralization tests (). Mice received 4 weekly intraperitoneal injections of 10% virus-infected newborn mouse brain suspension with Freud\xe2\x80\x99s adjuvant. Sarcoma 180 cells were also given intraperitoneally with a final injection to induce ascites formation. Antigenic differences among the viruses were then investigated by using cross-neutralization assays ()., In 1999, the virus strain IQT9646 was isolated from a 25-year-old male febrile patient who resided in Belen, Iquitos, Peru. The patient had an ' […]

Pipeline specifications

Software tools Trimmomatic, ABySS, Bowtie2, MEGA
Organisms Homo sapiens