Purifying the impure: sequencing metagenomes and metatranscriptomes from complex animal-associated samples

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• High-throughput sequencing has transformed biological research, necessitating advanced methods for analyzing complex host-associated microbial communities.
• Understanding viral and microbial communities is crucial for host health research, especially in challenging sample types like Cystic Fibrosis (CF) sputum.
• Existing methods often struggle with the viscosity and contaminants present in CF sputum, leading to inaccurate community profiling.

Purpose of the Study:

• To develop a comprehensive workflow for simultaneous DNA and RNA extraction from CF sputum.
• To generate viral and microbial metagenomes and metatranscriptomes from a single sample for next-generation sequencing.
• To optimize protocols for challenging sample types and assess the accuracy of the generated data.

Main Methods:

• A novel workflow was designed for concurrent DNA and RNA extraction, producing viral and microbial metagenomes and metatranscriptomes.
• The protocol specifically addresses challenges posed by CF sputum, including high viscosity, mucins, and free neutrophil DNA.
• Metatranscriptomics protocol was optimized to minimize ribosomal RNA (rRNA) contamination and recover both microbial and host mRNA.

Main Results:

• The workflow successfully recovered viral and microbial DNA from CF sputum with minimal human DNA contamination.
• Metatranscriptomics analysis yielded microbial and host mRNA with low rRNA content.
• Daily microbial profile fluctuations in CF patients were minimal without antibiotic treatment.
• Metagenomic and 16S ribosomal RNA gene sequencing showed similar taxonomic profiles for common CF bacteria when using hypotonic lysis (HL)-derived DNA.

Conclusions:

• The developed workflow provides a comprehensive approach to characterizing both the taxonomic and functional aspects of viral and microbial communities.
• Hypotonic lysis and washing steps are effective in removing human DNA and potentially misleading extracellular microbial DNA from CF sputum samples.
• This method offers a reliable tool for studying host-associated microbial communities in challenging clinical samples.