Non-invasive surveys of mammalian viruses using environmental DNA

Niccolo Alfano; Anisha Dayaram; Jan Axtner; Kyriakos Tsangaras; Marie-Louise Kampmann; Azlan Mohamed; Seth Timothy Wong; M. Thomas P. Gilbert; Andreas Wilting; Alex Daivd Greenwood

The RNA was reverse transcribed using SuperScript III and IV (Thermo Fisher Scientific) with random hexamers prior to second-strand synthesis with Klenow fragment (New England Biolabs). The resulting double-stranded cDNA/DNA mix was sheared to an average fragment size of 200 bp using a M220 focused ultrasonicator (Covaris). Sheared product was purified using the ZR-96 DNA Clean & Concentrator-5 kit (Zymo). Dual-indexed Illumina sequencing libraries were constructed as described by Meyer and Kircher 2010 [32] with the modifications described in Alfano et al. 2015 [33] . Each library was amplified in three replicate reactions to minimize amplification bias in individual PCRs. The three replicate PCR products for each sample were pooled and purified using the MinElute PCR Purification Kit (Qiagen). Negative control libraries were also prepared from different stages of the experimental process (extraction, reverse transcription, library preparation and index PCR) and indexed separately to monitor any contamination introduced during the experiment. Amplified libraries were quantified using the 2200 TapeStation (Agilent Technologies) on D1000 ScreenTapes.