Implications of error-prone long-read whole-genome shotgun sequencing on characterizing reference microbiomes

Abstract

20 21 total length 33,563,573,383 72,312,638,112 bases mapped 32,143,689,158 72,216,146,980 bases mapped (cigar) 31,156,025,998 70,073,211,829 mismatches 4,104,593,752 6,925,222,080 average length 4,150 3,961 maximum length 472,762 214,792 average Phred quality per base 13 17 157 The authors declare no conflict of interest. 595 596 623 SE, Cox SB, et al: Detecting Microbial Dysbiosis Associated with Pediatric Crohn Disease Despite 624 the High Variability of the Gut Microbiota. Cell Rep 2016, 14:945-955.

Recovery of complete genomes and non-chromosomal replicons from activated sludge enrichment microbial communities with long read metagenome sequencing

“…However substantial limitations of this approach have become evident, including problems related to the use of multi-sample co-assemblies 19,66,67 , the challenges of resolving genomes to strain level 68 , difficulties related to extracting MAGs from communities of high ecological complexity 69,70 and the limitations of automated binning procedures, requiring careful evaluation of recovered genomes 71 . In response to these challenges, recent efforts have combined short read with emerging complementary techniques such as HiC metagenomics [72][73][74] , synthetic long reads 75,76 , or long read sequencing 12,[16][17][18][19][20][21][22][23] and collectively these results suggest substantial improvements can be made in the quality and completeness of metagenome-assembled genomes using multiple types of sequence data.…”

“…We also show that while hybrid metagenome assembly generally did not recover complete chromosomal sequences, it does capture chromosomal length sequences that are not reconstructed from the use of long read data alone. Despite the fact that long read metagenome data collected from microbial communities of high to very high complexity are still emerging 18,20,22,23 , the analysis of datasets available to date suggests that binning procedures will have to be developed 18 or adapted from short read methods 20,22 , for effective recovery of genomes from such metagenome data.…”

“…New long read analysis methods 13,14 and binning algorithms designed for long read metagenome data 15 have also appeared, anticipating the future expansion of metagenome data generated from these new instruments. More recent studies 12,[16][17][18][19][20][21][22][23] have collectively established that full length (or near-full length genomes) can be recovered from long read sequencing of complex communities, which sets the stage for further development of genome-resolved long read metagenomics.…”

Long read metagenomics, the next step?

“…Taxonomy markers such as ribosomal RNA operons can be retrieved complete allowing a reliable community structure determination [21]. To assess the resolving power of the two most advanced LR sequencing available (Oxford Nanopore and PacBio Sequel II) and compare it with Illumina we have selected a real metagenome rather than artificial genome mixtures [22–24] or low diversity environments [25,26]. We still do not know the real extent of the diversity of a real-life complex community to be able to mimic it with mixtures of known genomes.…”

“…We still do not know the real extent of the diversity of a real-life complex community to be able to mimic it with mixtures of known genomes. Besides, this kind of test have already been done and provided satisfactory results [22–24]. The open ocean is one of the oldest and most important communities for the global ecology of the planet and has been extensively studied by several methods, including metagenomics, for decades [27–30].…”