Abstract
Shotgun metagenomics sequencing is a powerful tool for the characterization of complex biological matrices, enabling analysis of prokaryotic and eukaryotic organisms and viruses in a single experiment, with the possibility of reconstructing de novo the whole metagenome or a set of genes of interest. One of the main factors limiting the use of shotgun metagenomics on wide scale projects is the high cost associated with the approach. However, we demonstrate that—for some applications—it is possible to use shallow shotgun metagenomics to characterize complex biological matrices while reducing costs. We measured the variation of several summary statistics simulating a decrease in sequencing depth by randomly subsampling a number of reads. The main statistics that were compared are alpha diversity estimates, species abundance, detection threshold, and ability of reconstructing the metagenome in terms of length and completeness. Our results show that a classification of prokaryotic, eukaryotic and viral communities can be accurately performed even using very low number of reads, both in mock communities and in real complex matrices. With samples of 100,000 reads, the alpha diversity estimates were in most cases comparable to those obtained with the full sample, and the estimation of the abundance of all the present species was in excellent agreement with those obtained with the full sample. On the contrary, any task involving the reconstruction of the metagenome performed poorly, even with the largest simulated subsample (1M reads). The length of the reconstructed assembly was smaller than the length obtained with the full dataset, and the proportion of conserved genes that were identified in the meta-genome was drastically reduced compared to the full sample. Shallow shotgun metagenomics can be a useful tool to describe the structure of complex matrices, but it is not adequate to reconstruct—even partially—the metagenome.
Highlights
Shotgun metagenomics offers the possibility to assess the complete taxonomic composition of biological matrices and to estimate the relative abundances of each species in an unbiased way[1,2]
We provide more detail regarding the use of megahit for de novo assembly
Metagenome shotgun high-throughput sequencing has progressively gained popularity in parallel with the advancing of next-generation sequencing (NGS) technologies[3,4], which provide more data in less time at a lower cost than previous sequencing techniques
Summary
Shotgun metagenomics offers the possibility to assess the complete taxonomic composition of biological matrices and to estimate the relative abundances of each species in an unbiased way[1,2] It allows to agnostically characterize complex communities containing eukaryotes, fungi, bacteria and viruses. Metagenome shotgun high-throughput sequencing has progressively gained popularity in parallel with the advancing of next-generation sequencing (NGS) technologies[3,4], which provide more data in less time at a lower cost than previous sequencing techniques This allows the extensive application to study the most various biological mixtures such as environmental samples[5,6], gut samples[7,8,9], skin samples[10], clinical samples for diagnostics and surveillance purposes[11,12,13,14] and food ecosystems[15,16]. Several studies suggested that whole shotgun metagenome sequencing is more effective in the characterization of metagenomics samples compared to target amplicon approaches, with the additional capability of providing functional information regarding the studied approaches[23]
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