Abstract
BackgroundStudies of shifts in microbial community composition has many applications. For studies at species or subspecies levels, the 16S amplicon sequencing lacks resolution and is often replaced by full shotgun sequencing. Due to higher costs, this restricts the number of samples sequenced. As an alternative to a full shotgun sequencing we have investigated the use of Reduced Metagenome Sequencing (RMS) to estimate the composition of a microbial community. This involves the use of double-digested restriction-associated DNA sequencing, which means only a smaller fraction of the genomes are sequenced. The read sets obtained by this approach have properties different from both amplicon and shotgun data, and analysis pipelines for both can either not be used at all or not explore the full potential of RMS data.ResultsWe suggest a procedure for analyzing such data, based on fragment clustering and the use of a constrained ordinary least square de-convolution for estimating the relative abundance of all community members. Mock community datasets show the potential to clearly separate strains even when the 16S is 100% identical, and genome-wide differences is < 0.02, indicating RMS has a very high resolution. From a simulation study, we compare RMS to shotgun sequencing and show that we get improved abundance estimates when the community has many very closely related genomes. From a real dataset of infant guts, we show that RMS is capable of detecting a strain diversity gradient for Escherichia coli across time.ConclusionWe find that RMS is a good alternative to either metabarcoding or shotgun sequencing when it comes to resolving microbial communities at the strain level. Like shotgun metagenomics, it requires a good database of reference genomes and is well suited for studies of the human gut or other communities where many reference genomes exist. A data analysis pipeline is offered, as an R package at https://github.com/larssnip/microRMS.1VZ3xwdVv3tXqsbKN3Vx-QVideo abstract
Highlights
Studies of shifts in microbial community composition has many applications
We randomly selected 10 sequenced strains, and all results are based on retrieving the Reduced Metagenome Sequencing (RMS) fragments in silico from the genomes, using the cutting motifs GAATTC (EcoRI) and TTAA (MseI)
We notice that the number of RMS fragments per mega base pair varies a lot between species, but less within each species
Summary
Studies of shifts in microbial community composition has many applications. For studies at species or subspecies levels, the 16S amplicon sequencing lacks resolution and is often replaced by full shotgun sequencing. For communities like the human gut, extensive studies of the composition has given us the big picture, but recent investigations indicate that differences at the strain level may be crucial for phenotypic differences [5, 6] Common to these problems is the need for high-resolution taxonomic profiles that can be collected with moderate efforts and in a reproducible way. Such studies often require many samples in order to capture the biological variation, and since sequencing and computational resources are always limited, the simpler amplicon approach is often preferred to a deep shotgun sequencing in order to get enough samples covered. If separation at the species or strain level is required, the 16S marker is in general too conserved, and a full shotgun sequencing seems necessary
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