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

In-solution target enrichment via hybridization-based capture was performed according to the manufacturer's protocol (MYbaits® custom targeted enrichment, MYcroarray, Ann Arbor, USA), with the following modifications for likely partially degraded samples with an expected low target viral content: 50uL Dynabeads® M-270 Streptavidin beads (Invitrogen) instead of 30 uL Dynabeads® MyOne™ Streptavidin C1 (Invitrogen); hybridization, bead-bait binding, and wash steps temperature set to 60°C; 48 hours hybridization time; 200 ng baits per reaction; 10 μL indexed library inputs. For capture, libraries generated from pooled leeches consisting of more than 16 individuals were captured individually, while libraries generated from pools of fewer individuals were combined to have a comparable number (15) (16) (17) (18) (19) (20) of leeches per capture. This was done in order to ensure that each individual leech represented in each library was allocated enough bait for capture. For capture libraries generated from water and sediment samples. Samples were pooled in groups of two. Sediment and water cDNA and DNA were pooled separately. Per pooled sample, 5 µl of baits were used to ensure enough bait for each sample. The enriched libraries were reamplified using Herculase II Fusion DNA polymerase (Agilent Technologies) with P5 and P7 Illumina library outer primers with the same cycling conditions described in Alfano et al. 2016 . The re-amplified enriched libraries were purified using the MinElute PCR Purification Kit (Qiagen), quantified using the 2200 TapeStation (Agilent Technologies) on D1000 ScreenTapes and finally pooled in equimolar amounts for single-read sequencing on two lanes of an Illumina NextSeq 500 with the TG NextSeq® 500/550 High Output Kit v2 (300 cycles).