A restriction enzyme reduced representation sequencing approach for low-cost, high-throughput metagenome profiling

Abstract

Microbial community profiles have been associated with a variety of traits, including methane emissions in livestock. These profiles can be difficult and expensive to obtain for thousands of samples (e.g. for accurate association of microbial profiles with traits), therefore the objective of this work was to develop a low-cost, high-throughput approach to capture the diversity of the rumen microbiome. Restriction enzyme reduced representation sequencing (RE-RRS) using ApeKI or PstI, and two bioinformatic pipelines (referencebased and reference-free) were compared to bacterial 16S rRNA gene sequencing using repeated samples collected two weeks apart from 118 sheep that were phenotypically extreme (60 high and 58 low) for methane emitted per kg dry matter intake (n = 236). DNA was extracted from freeze-dried rumen samples using a phenol chloroform and bead-beating protocol prior to RE-RRS. The resulting sequences were used to investigate the repeatability of the rumen microbial community profiles, the effect of laboratory and analytical method, and the relationship with methane production. The results suggested that the best method was PstI RE-RRS analyzed with the reference-free approach, which accounted for 53.3±5.9% of reads, and had repeatabilities of 0.49±0.07 and 0.50±0.07 for the first two principal components (PC1 and PC2), phenotypic correlations with methane yield of 0.43±0.06 and 0.46±0.06 for PC1 and PC2, and explained 41±8% of the variation in methane yield. These results were significantly better than for bacterial 16S rRNA gene sequencing of the same samples (p<0.05) except for the correlation between PC2 and methane yield. A Sensitivity study suggested approximately 2000 samples could be sequenced in a single lane on an Illumina HiSeq 2500, meaning the current work using 118 samples/lane and future proposed 384 samples/lane are well within that threshold. With minor adaptations, our approach could be used to obtain microbial profiles from other metagenomic samples.

Reduced metagenome sequencing for strain-resolution taxonomic profiles

“…We believe our results indicate the RMS approach for metagenome profiling is something to explore further. We have focused a lot on one pair of restriction enzymes in this study, but other enzymes are used for similar studies [ 7 , 9 ]. The choice of enzymes will affect the number and length of fragments, but apart from this, the data analysis procedure we propose here may be used, as we illustrate by the reanalysis of the data from [ 7 ].…”

“…As an alternative to do a full shotgun metagenomic sequencing, the use of restriction enzymes to reduce the genomic sequence space has also been employed to investigate microbial communities [ 7 – 9 ]. The double-digested restriction-associated DNA sequencing (ddRADseq) idea [ 10 – 12 ] has been along for some time, but its use for metagenome studies is quite new.…”

“…Also, due to the variable lengths and compositions of the fragments, the PCR-amplification efficiency must be expected to vary and create biases. For purely predictive purposes, such reference-free approaches may still be useful, as suggested by [ 9 ].…”

Large-Scale Phenotyping of Livestock Welfare in Commercial Production Systems: A New Frontier in Animal Breeding

“…Targeted sequencing and metagenome shotgun sequencing are the two main approaches for generating microbiome profiling. Recently, a low-cost and high-throughput approach based on Restriction-Enzyme Reduced Representation Sequencing (RE-RRS) has been proposed as an alternative to capture the diversity of the rumen microbiome (Hess et al, 2020). As the costs to generate sequencing datasets decrease, microbiome profiling might be an additional relevant phenotype for further investigations and potential applications for selection to improved welfare in livestock species.…”

The potential for mitigation of methane emissions in ruminants through the application of metagenomics, metabolomics, and other -OMICS technologies

“…Metagenomic profiling has been applied to microbiome analysis in ruminant research, such as the characterization of the rumen microbial metagenome and its association with performance traits ( Carberry et al, 2014a , b ; Kamke et al, 2016 ; Roehe et al, 2016 ; Wang et al, 2017 ; Hess et al, 2020 ). A sophisticated network of symbiotic relationships exists between the (rumen) microbiota and the host, which are essential for metabolic maintenance, immune function, and overall production efficiency of the host ( Hobson, 1997 ).…”