NGS Library Construction for Microbiome Studies

Contributed by Carol Huang

Studying the population composition of microbes within a particular environmental community has drawn more and more attention for understanding functions and interactions of microbial communities.

Meta-RNA-Sequencing and Meta-Genomic-sequencing have been playing big roles in these studies. As a part of the study, I have been focusing on library construction. There have been many challenges in the process. The first one is sample types, like high-fat content, other chemical residues from sample collecting sources, and host proportion. It’s not easy to completely remove fat during meta-RNA and meta-DNA isolation, which would affect RNA/ DNA purity and would interfere with the downstream process, as would chemical residues.  It’s easier for RNA / DNA to be released from some types of host cells than those from microbes. If there is a big portion of the host in the sequencing libraries, the sequencing coverage for microbes would be decreased. The second one is the abundance of microbes in different sample types.  The third is to make maximum inclusive, representative sequencing libraries.   Decision making during library construction, such as fragmentation and size selection is critical, for these process might get rid of some microbe populations. That would make the sequencing data less inclusive of all possible microbe populations in the community.

Every sample type is a new challenge for me, but over time I have conquered challenges one-by-one and learned quite a lot during these practices. We have made a lot of population inclusive meta-RNA sequencing libraries from pet food, fat tissue, feces, and others.

There are many new challenges every day waiting for us to explore, comprehend, and conquer.

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