Enhanced Recovery of Microbial Genes and Genomes From a Marine Water Column Using Long-Read Metagenomics

Abstract

Third-generation sequencing has penetrated little in metagenomics due to the high error rate and dependence for assembly on short-read designed bioinformatics. However, second-generation sequencing metagenomics (mostly Illumina) suffers from limitations, particularly in the assembly of microbes with high microdiversity and retrieval of the flexible (adaptive) fraction of prokaryotic genomes. Here, we have used a third-generation technique to study the metagenome of a well-known marine sample from the mixed epipelagic water column of the winter Mediterranean. We have compared PacBio Sequel II with the classical approach using Illumina Nextseq short reads followed by assembly to study the metagenome. Long reads allow for efficient direct retrieval of complete genes avoiding the bias of the assembly step. Besides, the application of long reads on metagenomic assembly allows for the reconstruction of much more complete metagenome-assembled genomes (MAGs), particularly from microbes with high microdiversity such as Pelagibacterales. The flexible genome of reconstructed MAGs was much more complete containing many adaptive genes (some with biotechnological potential). PacBio Sequel II CCS appears particularly suitable for cellular metagenomics due to its low error rate. For most applications of metagenomics, from community structure analysis to ecosystem functioning, long reads should be applied whenever possible. Specifically, for in silico screening of biotechnologically useful genes, or population genomics, long-read metagenomics appears presently as a very fruitful approach and can be analyzed from raw reads before a computationally demanding (and potentially artifactual) assembly step.

Long-Read Metagenomics Improves the Recovery of Viral Diversity from Complex Natural Marine Samples

“…This sample was sequenced with Illumina and PacBio Sequel II systems and then assembled twice, first using only the Illumina short reads, resulting in the short-read assembly data set (SRa), and then in a hybrid assembly using both the Illumina short reads and the PacBio long reads, resulting in the long-read assembly data set (LRa). We decided on the hybrid assembly rather than a long-read-only assembly based on previous results ( 12 ). In order to evaluate the possible biases introduced by the assembly process, we also analyzed the PacBio CCS15 reads (PacBio consensus reads created by comparing at least 15 subreads [LR]) before assembly.…”

“…This bias could arise from the fact that assemblies usually recover only the core genome. In this sample (marine surface water), clade SAR11 is the most abundant organism ( 12 ), with an average GC content of 34%. LRs recover more of the flexible genome, which can present GC fluctuations compared to the core and would thus explain this variation from 34% to 38%.…”

“…The development of high-fidelity approaches such as PacBio circular consensus sequencing (CCS) with an error rate similar to that of the Illumina system opens a new avenue for the study of prokaryotic communities in their natural environments. An example of the advantages of this new technology can be found in a recent work, in which a well-known marine sample from the Mediterranean water column was analyzed using both SR and LR sequencing ( 12 ). Results suggested that PacBio Sequel II CCS is particularly suitable for cellular metagenomics due to its large read size and its low error rate.…”

“…Reads in LR metagenomes are large enough to perform gene prediction directly, bypassing the biases inherent in the assembly process. The assembly step is also improved with this kind of sample by using hybrid assembly of LR and SR, allowing reconstruction of genomes, including the flexible genome and even streamlined genomes, such as those from Pelagibacterales ( 12 ).…”

Phylogenomics of SAR116 Clade Reveals Two Subclades with Different Evolutionary Trajectories and an Important Role in the Ocean Sulfur Cycle

“…The example of SAR116 is not unique since other examples of processes with the same trend have already been described. In most of these cases, single-cell genomics is revealing new lineages of microbes with streamlined genomes that are very abundant in nature but difficult to obtain in pure culture ( 46 , 48 , 73 ). In the case of the marine Roseobacter clade, the use of single-cell genomics has allowed for the description of uncultivated streamlined lineages that together represent ca.…”