Completion of draft bacterial genomes by long-read sequencing of synthetic genomic pools

Abstract

Background: Illumina technology currently dominates bacterial genomics due to its high read accuracy and low sequencing cost. However, the incompleteness of draft genomes generated by Illumina reads limits their application in comprehensive genomics analyses. Alternatively, hybrid assembly using both Illumina short reads and long reads generated by single molecule sequencing technologies can enable assembly of complete bacterial genomes, yet the high per-genome cost of long-read sequencing limits the widespread use of this approach in bacterial genomics. Here we developed a protocol for hybrid assembly of complete bacterial genomes using miniaturized multiplexed Illumina sequencing and non-barcoded PacBio sequencing of a synthetic genomic pool (SGP), thus significantly decreasing the overall per-genome cost of sequencing. Results: We evaluated the performance of SGP hybrid assembly on the genomes of 20 bacterial isolates with different genome sizes, a wide range of GC contents, and varying levels of phylogenetic relatedness. By improving the contiguity of Illumina assemblies, SGP hybrid assembly generated 17 complete and 3 nearly complete bacterial genomes. Increased contiguity of SGP hybrid assemblies resulted in considerable improvement in gene prediction and annotation. In addition, SGP hybrid assembly was able to resolve repeat elements and identify intragenomic heterogeneities, e.g. different copies of 16S rRNA genes, that would otherwise go undetected by short-read-only assembly. Comprehensive comparison of SGP hybrid assemblies with those generated using multiplexed PacBio long reads (long-read-only assembly) also revealed the relative advantage of SGP hybrid assembly in terms of assembly quality. In particular, we observed that SGP hybrid assemblies were completely devoid of both small (i.e. single base substitutions) and large assembly errors. Finally, we show the ability of SGP hybrid assembly to differentiate genomes of closely related bacterial isolates, suggesting its potential application in comparative genomics and pangenome analysis. Conclusion: Our results indicate the superiority of SGP hybrid assembly over both short-read and long-read assemblies with respect to completeness, contiguity, accuracy, and recovery of small replicons. By lowering the pergenome cost of sequencing, our parallel sequencing and hybrid assembly pipeline could serve as a cost effective and high throughput approach for completing high-quality bacterial genomes.

Identification and Quantification of Bovine Digital Dermatitis-Associated Microbiota across Lesion Stages in Feedlot Beef Cattle

“…DNA was extracted from these isolates using the Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, and normalized to 5 ng/μl using the Qubit dsDNA (double-stranded DNA) HS kit (Life Technologies, Carlsbad, CA, USA). Sequencing and draft genome assembly were conducted as described previously by Derakhshani et al ( 40 ) for barcoded Illumina HiSeq reads. Briefly, sequencing libraries were prepared using the NEBNext Ultra II FS DNA library prep kit for Illumina (New England BioLabs [NEB], Ipswich, MA, USA).…”

Next Generation Microbiome Research: Identification of Keystone Species in the Metabolic Regulation of Host-Gut Microbiota Interplay

“…A more recent study showed that the use of long-read technologies (e.g., PacBio) improved drastically the completeness of the MAGs and associated predicted genes: more than 98% of the genes predicted from PacBio-assembled MAGs were complete compared to only 40% using HiSeq-assembled MAGs ( Xie et al, 2020 ). Indeed, the use of long reads enables the detection of repeated elements often found in ribosomal RNA genes or bacteriophage-related insertions that are frequently missed with short read sequencing ( Derakhshani et al, 2020 ).…”

Novel insights into the role of the mobilome in ecological diversification and success of Staphylococcus haemolyticus as an opportunistic pathogen

“…As the study was based on draft genome sequences, it is challenging to study repetitive elements like insertion sequence (IS) elements and other mobile elements such as plasmids integrons, CRISPRs, conjugative transposons, and phages. With the advent of third-generation sequencing, it is possible to obtain complete genome sequences to understand intragenomic heterogeneity and inter-strain variations in an effective manner [14]. In the present study, we provide evidence for genome expansion and the role of specific IS elements along with plasmids in the ecological diversification of S. haemolyticus.…”