Search

Environmental DNA (eDNA) sampling is an established tool for biodiversity studies. The design of assays to detect species’ DNA in samples underpins this tool. Although much progress has been made to develop validation frameworks for assay performance, an in silico validation framework is sorely missing. A conceptual framework was developed to incorporate the elements of assay design and likely outcomes of assay performance through a step-by-step decision tree evaluating in silico sensitivity and specificity. Using an in silico PCR tool, the sensitivity and specificity of published assays were evaluated to determine the potential ability of an assay to amplify and detect known target and non-target species sequences and place them within the conceptual framework. Results showed that most published assays tested exhibited the potential for Type I and/or Type II errors, with very few assays showing unique species specificity. Results also demonstrate that in silico validation can predict potential problematic non-target species co-amplification. The developed conceptual framework demonstrates the ability to provide a robust and reproducible in silico validation step highlighting key components of the design stage that need to be published to improve transparency, repeatability, reproducibility, and confidence in eDNA sampling.

This article is free to read on the publishers website The nucleotide sequence of a cellulase cDNA (celA) from the rumen fungus Neocallimastix patriciarum and the primary structure of the protein which it encodes were characterized. The celA cDNA was 1.95 kb long and had an open reading frame of 1,284 bp, which encoded a polypeptide having 428 amino acid residues. A sequence alignment showed that cellulase A (CELA) exhibited substantial homology with family B cellulases (family 6 glycosyl hydrolases), particularly cellobiohydrolase II from the aerobic fungus Trichoderma reesei. In contrast to previously characterized N. patriciarum glycosyl hydrolases, CELA did not exhibit homology with any other rumen microbial cellulases described previously. Primary structure and function studies in which deletion analysis and a sequence comparison with other well-characterized cellulases were used revealed that CELA consisted of a cellulose-binding domain at the N terminus and a catalytic domain at the C terminus. These two domains were separated by an extremely Asn-rich linker. Deletion of the cellulose-binding domain resulted in a marked decrease in the cellulose-binding ability and activity toward crystalline cellulose. When CELA was expressed in Escherichia coli, it was located predominantly in the periplasmic space, indicating that the signal sequence of CELA was functional in E.coli. Enzymatic studies showed that CELA had an optimal pH of 5.0 and an optimal temperature of 40 degrees C. The specific activity of immunoaffinity-purified CELA against Avicel was 9.7 U/mg of protein, and CELA appeared to be a relatively active cellobiohydrolase compared with the specific activities reported for other cellobiohydrolases, such as T. reesei cellobiohydrolases I and II.