Metagenomic analysis of the RNA fraction of the fecal virome indicates high diversity in pigs infected by porcine endemic diarrhea virus in the United States

Chen, Qi; Wang, Leyi; Zheng, Ying; Zhang, Jianqiang; Guo, Baoqing; Yoon, Kyoung-Jin; Gauger, Phillip C.; Harmon, Karen M.; Main, Rodger G.; Li, Ganwu

The output of sequence data was demultiplixed based on default setting of MiSeq. The primary workflow used to identify mixed infections is outlined in Fig. 1 . The default parameters were used to perform analysis on all the softwares used in the study. Raw sequencing reads from MiSeq were subjected to sequencing quality analysis with FastQC pre-installed in the Illumina MiSeq® instrument with defaulting parameter setting. Reads with low sequencing quality, sequencing adaptors and primers were removed from further bio-informative analysis with Trimmomatic v0.36 [10] . Cleaned reads were classified with the standard database Kraken v0.10.5-beta including pig host, refseq virus, bacteria, and archaea sequences ( Fig. 1 ) [11] . KronaTools-2.6 was then used to generate the interactive charts for the hierarchical classification results and any classified organisms can be viewed in these pie charts [12] . To confirm the classification and to control the quality of assembling results of Kraken method, Kaiju program version 1.6.2 was used as a secondary validating method, which determines taxonomic classification on protein level [13] .