Emerging infectious diseases in humans are frequently caused by pathogens originating from animal hosts, and zoonotic disease outbreaks present a major challenge to global health. To investigate drivers of virus spillover, we evaluated the number of viruses mammalian species have shared with humans. We discovered that the number of zoonotic viruses detected in mammalian species scales positively with global species abundance, suggesting that virus transmission risk has been highest from animal species that have increased in abundance and even expanded their range by adapting to human-dominated landscapes. Domesticated species, primates and bats were identified as having more zoonotic viruses than other species. Among threatened wildlife species, those with population reductions owing to exploitation and loss of habitat shared more viruses with humans. Exploitation of wildlife through hunting and trade facilitates close contact between wildlife and humans, and our findings provide further evidence that exploitation, as well as anthropogenic activities that have caused losses in wildlife habitat quality, have increased opportunities for animal-human interactions and facilitated zoonotic disease transmission. Our study provides new evidence for assessing spillover risk from mammalian species and highlights convergent processes whereby the causes of wildlife population declines have facilitated the transmission of animal viruses to humans.

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.