Host DNA depletion on frozen human respiratory samples enables successful metagenomic sequencing for microbiome studies

Abstract

Background: Most respiratory microbiome studies have focused on amplicon rather than metagenomics sequencing due to high host DNA content. We evaluated efficacy of five host DNA depletion methods on previously frozen human bronchoalveolar lavage (BAL), nasal swabs, and sputum prior to metagenomic sequencing.

Results: Median sequencing depth was 76.4 million reads per sample. Untreated nasal, sputum and BAL samples had 94.1%, 99.2%, and 99.7% host-reads. The effect of host depletion differed by sample type. Most treatment methods increased microbial reads, species richness and predicted functional richness; the increase in species and predicted functional richness was mediated by higher effective sequencing depth. For BAL and nasal samples, most methods did not change Morisita-Horn dissimilarity suggesting limited bias introduced by host depletion.

Conclusions: Metagenomics sequencing without host depletion will underestimate microbial diversity of most respiratory samples due to shallow effective sequencing depth and is not recommended. Optimal host depletion methods vary by sample type.

Keywords: Microbiome; Respiratory; Shotgun metagenomics sequencing; host DNA depletion; low biomass.

Figure 1. Overview of study design.

Samples collected from the same participant were aliquoted so that paired comparisons could be made between treated and untreated samples. For nasal samples, it was only feasible to collect 4 swabs from a participant at the same time, thus a total of 10 swabs for the untreated condition was required to allow these paired treated and untreated comparisons.

Figure 2. Sample relative read abundances at the species level stratified by sample type and host depletion method.

2A Broncho-alveolar lavage (BAL) from critically ill patients. 2BNasal swab samples from healthy adults. 2C Spontaneously expectorated sputum from people living with cystic fibrosis. Empty space indicates samples that failed sequencing (no microbial reads identified).

Figure 3. Alpha and beta diversity stratified by sample type and treatment method.

3A.Species richness in mean values ± SD. 3B. Boxplot of potential bias measured by Morisita-Horn dissimilarity (1 – MH) between each host depletion method and corresponding untreated sample. Statistical significances were tested with linear mixed effect model adjusting for repeated measures in a participant as a random effect variable. *: p-value < 0.05, **: p-value < 0.01 and ***: p-value < 0.001.

Figure 4

Mean relative abundance of top 20 significant taxa identified by differential abundance analysis using linear mixed effect model (feature ~ lyPMA + Benzonase + HostZero + MolYsis + QIAamp + (1|subject id)) after centered log-ratio transformation. Analyses were stratified by sample type. (A) Bronchoalveolar lavage, (B) Nasal, and (C) Sputum. Statistical significances were noted at the level of q-value < 0.1.