Cbot workstation

Poly-A+ RNA was selected from total RNA (1 μg) using oligo dT-attached magnetic beads. Bound RNA was fragmented by incubation at 94 °C for eight (8) minutes in 19.5 μl of fragmentation buffer (Illumina). First and second strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq RNA Sample Preparation kit under conditions prescribed by the manufacturer (Illumina). Samples were tracked using “index tags” incorporated into the adapters. Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantification of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Template input was adjusted to obtain a cluster density of 700–900 K/mm2. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000 using protocols defined by the manufacturer.

Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Template input was adjusted to obtain a cluster density of 700–850 K/mm². 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000 using protocols defined by the manufacturer.

Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) 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) on an Illumina HiSeq 1000 using protocols defined by the manufacturer. Cluster density per lane was 645–980 k/mm² and post filter reads ranged from 148–178 million per lane. Base call conversion to sequence reads wasperformed using CASAVA-1.8.2. Virus sequences were edited and assembled using the SeqMan and NextGen modules of the DNAStar Lasergene 7 program (Bioinformatics Pioneer DNAStar, Inc., Madison, WI). In certain cases, prefiltering of reads to remove host sequence enhanced the assembly process. Assembly was carried out using a fasta file of Aedes albopictus sequences to remove host DNA from the assembly thus reducing the number of contigs present.

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)].

Viral RNA (0.05–1.7 mg) was fragmented by incubation at 94°C for 8 minutes in 19.5 μl of fragmentation buffer (Illumina, San Diego, CA). First and second strand synthesis, adapter ligation, and amplification of the library were performed using the Illumina TruSeq RNA Samplec Preparation kit as per the manufacturer’s protocol. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation as per the manufacturer’s protocol. Paired-end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1500 as per the manufacturer’s protocol.
All reads were assembled using a pipeline previously described [7 (link)] and were assembled using the Venezuelan equine encephalitis virus strain 68U201 (GenBank accession #: U34999.1; [35 (link)]) as a reference sequence. Diversity was calculated using Shannon entropy [36 ] and a cut off of 1% was used for the analysis.