Analysis of intrapatient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus

Park, Donghyun; Huh, Hee Jae; Kim, Yeon Jeong; Son, Dae-Soon; Jeon, Hyo-Jeong; Im, Eu-Hyun; Kim, Jong-Won; Lee, Nam Yong; Kang, Eun-Suk; Kang, Cheol In; Chung, Doo Ryeon; Ahn, Jin-Hyun; Peck, Kyong Ran; Choi, Sun Shim; Kim, Yae-Jean; Ki, Chang-Seok; Park, Woong-Yang

The PCR products from each patient were combined into a pool of approximately equal molarity and subjected to paired-end library construction with a TruSeq Nano DNA Sample prep kit (Illumina). Libraries were sequenced using an Illumina MiSeq sequencer (Illumina), generating 150-bp paired-end reads. On average, 4113× and 9361× raw read depths were achieved for full-genome and targeted deep sequencing, respectively. Sequencing metrics including mean depth of coverage of each sample are summarized in Supplemental Table S7 . Bases were called with MiSeq reporter software (ver. 2.4.60). The reads were quality filtered using the command "fastq_quality_filter," which required a minimum of 90% bases with a quality score of 20 or higher. Paired-end reads were aligned with the National Center for Biotechnology Information (NCBI) MERS-CoV reference sequence (NC_019843.3) using the Burrows-Wheeler Aligner (BWA) (Li and Durbin 2010) . SAMtools v0.1.19 (Li et al. 2009 ), GATK v2.4-7 (McKenna et al. 2010 , and Picard v1.93 were used for sorting SAM/BAM files, local realignment, and duplicate markings, respectively. We used the SAMtools "mpileup" command to determine the read depth. The consensus sequence was determined from the most commonly expressed nucleotide at each position.