Sequence-Independent Single Primer Amplification for Viral Discovery

Sequences were assembled using sequence-independent single primer amplification (SISPA) to barcode random primed cDNAs [29 (link), 30 (link)] from individual cDNA samples. SISPA products were normalized and pooled into a single reaction that was purified using a PCR purification kit (Qiagen, Valencia, CA). Samples were subsequently gel purified to select for products ranging from 300-500bp in size for sequencing with the Illumina Genome Analyzer II or 500-800bp in size for Roche 454 Titanium (GS-FLX) sequencing [31 (link)], or were sequenced on the Illumina HiSeq using the following protocol; cDNA (0.05–1.7 μg) was fragmented by incubation at 94°C for eight (8) minutes in 19.5 ul of fragmentation buffer (Illumina 15016648). Samples were tracked using the “index tags” incorporated into the adapters as 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 1500 using protocols defined by the manufacturer.