CDCap™ Ultra-Comprehensive Pathogen Kit

Support. Different library preparation approaches can be employed for RNA and DNA viruses. One approach involves the concurrent library preparation of RNA and DNA viruses, followed by subsequent hybridization capture steps. Another approach entails preparing RNA libraries and DNA libraries separately, and then mixing them for hybridization capture. Generally, for RNA libraries, there is no need to perform the step of removing of host ribosomal RNA.

Due to significant variations in pathogen content among different samples, if the aim is to achieve similar data amounts for each sample, it’s not feasible to perform multiple-plex hybridization; separate hybridization must be carried out instead. When there are orders of magnitude differences in microbial content among samples, although there are substantial variations in data amounts, low-abundance samples require relatively less data on their own. Moreover, different samples, using the same experimental parameters, exhibit similar PCR duplication rates. If they are subjected to separate hybrid capture and provided with the same sequencing data, low-abundance samples would need an increased number of PCR cycles. The identical information content would only be repeated to form an augmented sequencing data, as post-panel capture sequencing becomes easier to saturate. Based on these considerations, for samples with similar origins, performing the multiple-plex hybridization process is recommended.