RNA-Seq From Low-Ct RT-qPCR Samples

For ten samples with low Ct values in RT-qPCR assays, viral RNA was extracted then fragmented by incubation at 94°C for eight (8) minutes in 19.5 μl of fragmentation buffer (Illumina Inc., San Diego, CA). First and second strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq RNA Sample Preparation kit v2 under conditions prescribed by the manufacturer (Illumina Inc., San Diego, CA). Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina Inc., San Diego, CA) and the Illumina cBot workstation using conditions recommended by the manufacturer. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina Inc., San Diego, CA) on an Illumina HiSeq 1000 using protocols defined by the manufacturer.
Cluster density per lane was 820–940 k/mm² and post-filter reads ranged from 148–218 million per lane. Base call conversion to sequence reads was performed using CASAVA-1.8.2. Reads were filtered for quality and adapter sequences were removed, then viral contigs were assembled de novo using AbySS software [24 (link)]. Assembled contigs were checked using bowtie2 to align reads to the contigs [25 (link)] followed by visualization using the integrative genomics viewer [26 (link)].