An optimized approach for processing of frozen lung and lavage samples for microbiome studies

Abstract

The respiratory tract has a resident microbiome with low biomass and limited diversity. This results in difficulties with sample preparation for sequencing due to uneven bacteria-to-host DNA ratio, especially for small tissue samples such as mouse lungs. We compared effectiveness of current procedures used for DNA extraction in microbiome studies. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected to test different forms of sample pre-treatment and extraction methods to increase bacterial DNA yield and optimize library preparation. DNA extraction using a pre-treatment method of mechanical lysis (lung tissue) and one-step centrifugation (BALF) increased DNA yield and bacterial content of samples. In contrast, a significant increase of environmental contamination was detected after phenol chloroform isoamyl alcohol (PCI) extraction and nested PCR. While PCI has been a standard procedure used in microbiome studies, our data suggests that it is not efficient for DNA extraction of frozen low biomass samples. Finally, a DNA Enrichment kit was tested and found to improve the 16S copy number of lung tissue with a minor shift in microbial composition. Overall, we present a standardized method to provide high yielding DNA and improve sequencing coverage of low microbial biomass frozen samples with minimal contamination.

Fig 1. Workflow for processing BALF and lung samples for microbiome data.

Initially we compared two DNA extraction methods (experimental kit-based and PCI) and three forms of sample pre-treatment method. A small subset of samples was used to compare the efficiency of Microbial Enrichment kit. The V4 16S region was selected for preparing two nested (25x25 and 35x25 PCR cycles) and one non-nested (35 PCR cycles) libraries preparation methods.

Fig 2. DNA content and estimated biomass of BALF samples.

A) DNA concentration (ng/μL) of BALF samples, B) total read count is estimated based on the 16S rRNA gene copy number after quality filtering of the data, C) estimated DNA content dividing total DNA extracted by volume of the sample processed, and D) estimated bacterial DNA content dividing read count by volume of the sample processed. Significance (p-value) between groups is indicated by stars (* <0.05 and ** < 0.01). Based on overall data, most effective pre-treatment method for BALF samples was a one Method centrifugation followed by mechanical cell lysis. Two Method centrifugation was less effective.

Fig 3. DNA content and estimated biomass of lung tissue samples.

A) DNA concentration (ng/μL) of lung tissue samples, B) total read count is estimated based on the 16S rRNA gene copy number after quality filtering of the data, C) estimated DNA content dividing total DNA extracted by weight of the sample processed, and D) estimated bacterial DNA content dividing read count by weight of the sample processed. Significance (p-value) between groups is indicated by stars (* <0.05, ** < 0.01, *** < 0.001, and **** < 0.0001). Overall, most mechanically lysed tissue samples resulted in high DNA yield with exception of the PCI treated samples.

Fig 4. Diversity analysis of BALF and lung tissue samples.

A) Bacterial composition of lung and BALF samples. Overall, samples had a higher abundance of Proteobacteria and Firmicutes bacteria. B) Alpha diversity measures shows that BALF samples have a higher species richness compared to both lavaged and unlavaged lung samples. Observed significant (p < 0.05) difference in species richness between BALF and lavaged lung samples. C) Beta diversity plot shows admixture of sample types. D) Statistical summary of ADONIS test shows the variance and significance when comparing mice age and extraction pre-treatment methods.

Fig 5. Comparing the efficiency before and after treatment with the NEBNext microbiome DNA Enrichment Kit.

A) No significant improvements observed when comparing microbiome enriched (ME) and non-treated (NT) BALF samples total read count. B) However, significant improvements observed when comparing DME (1:10 diluted sample then treated with ME kit), ME, and NT lung samples total read count. Significance (p-value) between groups is indicated by stars (* <0.05 and ** < 0.01). Both C) BALF and D) lung samples showed difference in community composition (genus level) between NT and ME samples, particularly diluted lung samples treated with microbiome enrichment kit.