title: genome sequencing of Sindbis virus experimental  population
design: Viral RNA was isolated using the QIAamp Viral RNA mini kit (Qiagen). All subsequent steps, from reverse transcription through sequencing, were completed in duplicate, resulting in two technical replicates of each sample (entered here as ÔAŐ and ÔBŐ). cDNA was generated by reverse-transcription with Superscript II (Life Technologies) using random hexamer primers. The entire genome sequence was amplified via PCR with GoTaq DNA Polymerase (Promega) using eight primer pairs, generating overlapping PCR fragments 1.5-2.1 kb in length. Amplified genome fragments were purified using the QIAquick PCR Purification Kit (Qiagen), and the eight fragments from each virus sample were combined to create an equamolar mixture. Libraries were prepared from the pooled viral amplicons using the Nextera XT Kit (Illumina Inc.) and the Nextera Index Kit (Illumina Inc.). Samples were sequenced in a single lane via paired-end, 75-bp read Illumina HiSeq 2500 (Illumina Inc.) at the Yale Center for Genome Analysis.