Biology subjects

Frey, J. E.

Publications and source records attributed to Frey, J. E..

5 recordsLinked to original sources

bioRxiv2026

GenomeCompendium: A database for the integrated analysis of repeats, assembly quality and functional content of complete prokaryotic genomes

Microorganisms hold great promise for urgent global needs such as increasing sustainable agricultural production while reducing chemical fertilizer and pesticide use or providing novel classes of antimicrobials/therapeutics. Moving from analyzing microbiome composition to applying synthetic communities and studying their functions requires access to isolates and complete genome sequences. By spanning the frequent repeats, long-read sequencing can resolve complex prokaryotic genomes, yet error-prone short-read assemblies dominate. We here release the GenomeCompendium, a public database and interactive analysis tool for complete prokaryotic genomes (https://genome-compendium.com/). Using NCBI RefSeq (~47,000) and GenBank (~13,000) genomes, we integrated available metadata, GTDB taxonomy and computed features including repeat classes and gene content screening, intragenomic 16S rRNA sequence identity, and biosynthetic gene cluster co-occurrences. Evaluating repeat content and assembly complexity metrics, we identify taxonomic ranks dominated by difficult-to-assemble genomes and show that complex, repeat-rich genomes are more common than previously estimated. By mining metadata, our quality control flags 6.3% of RefSeq assemblies as potentially erroneous or incomplete. As valuable reference for data mining and to track taxonomic coverage, the GenomeCompendium links ~90 features across genomes, offers downloadable reports and -as unique features- pre-computed proteogenomics databases to improve genome annotations of RefSeq strains and the ability to analyze any uploaded prokaryotic genome.

genomics↗

bioRxiv2024

Chromosome-level haplotype-resolved genome assembly provides insights into the highly heterozygous genome of Italian ryegrass (Lolium multiflorum Lam.)

Italian ryegrass (Lolium multiflorum Lam.) is an important forage grass, providing a major source of feed for ruminants in temperate regions. Due to its highly heterozygous and repeat-rich genome, high-quality chromosome-level genome assemblies are scarce for Italian ryegrass. Here, we sequenced the genome of a genotype from the Italian ryegrass cultivar Rabiosa (hereafter referred to as Rabiosa), and we obtained Oxford Nanopore Technologies long reads ([~]60-fold coverage), Illumina short reads ([~]85-fold coverage) and high-throughput chromosome conformation capture data ([~]60-fold coverage). With Rabiosa as the parent, we constructed an F1 population consisting of 305 individuals, which were genotyped by reduced representation sequencing for linkage map construction and quantitative trait loci (QTL) analysis. Using whole-genome sequencing data of Rabiosa and the genetic linkage map, we first generated a chromosome-level unphased haploid assembly (scaffold N50 of 338.75 Mb, total BUSCO score of 94.60%). Then, based on the unphased assembly and a reference-based phasing approach, we generated a chromosome-level haplotype-resolved assembly containing both haplotypes (scaffold N50 of [~]250 Mb and total BUSCO score of [~]90% for each haplotype). Between the two haplotypes of Rabiosa, we observed a highly collinear gene order at chromosome level and a high sequence variation at local level. With a graph-based reference built from the unphased and the haplotype-resolved assemblies of Rabiosa, we conducted a QTL analysis, and two QTL significantly associated with stem rust resistance were detected. The genome assemblies of Rabiosa will serve as invaluable genomic resources to facilitate genomic applications in forage grass research and breeding.

genomics↗

bioRxiv2018

Long read-based de novo assembly of low complex metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system

BackgroundComplete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled-genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been shown so far. Natural whey starter cultures (NWCs) are used in the production of cheese and represent low complex microbiomes. Previous studies of Swiss Gruyere and selected Italian hard cheeses, mostly based on amplicon-based metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii.\n\nResultsTwo NWCs from Swiss Gruyere producers were subjected to whole metagenome shotgun sequencing using Pacific Biosciences Sequel, Oxford Nanopore Technologies MinION and Illumina MiSeq platforms. We achieved the complete assembly of all dominant bacterial genomes from these low complex NWCs, which was corroborated by a 16S rRNA based amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial genomes, we could also assemble several bacterial plasmids as well as phages and a corresponding prophage. Biologically relevant insights could be uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing third generation, long-read sequencing data able to span intragenomic as well as intergenomic repeats.\n\nConclusionsHere, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low complex NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omic analyses, functional profiling and phenotype to genotype analysis of specific microbial communities.

genomics↗

bioRxiv2018

Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats

Generating a complete, de novo genome assembly for prokaryotes is often considered a solved problem. However, we here show that Pseudomonas koreensis P19E3 harbors multiple, near identical repeat pairs up to 70 kilobase pairs in length. Beyond long repeats, the P19E3 assembly was further complicated by a shufflon region. Its complex genome could not be de novo assembled with long reads produced by Pacific Biosciences technology, but required very long reads from the Oxford Nanopore Technology. Another important factor for a full genomic resolution was the choice of assembly algorithm.\n\nImportantly, a repeat analysis indicated that very complex bacterial genomes represent a general phenomenon beyond Pseudomonas. Roughly 10% of 9331 complete bacterial and a handful of 293 complete archaeal genomes represented this dark matter for de novo genome assembly of prokaryotes. Several of these dark matter genome assemblies contained repeats far beyond the resolution of the sequencing technology employed and likely contain errors, other genomes were closed employing labor-intense steps like cosmid libraries, primer walking or optical mapping. Using very long sequencing reads in combination with assemblers capable of resolving long, near identical repeats will bring most prokaryotic genomes within reach of fast and complete de novo genome assembly.

genomics↗

bioRxiv2017

An integrative strategy to identify the entire protein coding potential of prokaryotic genomes by proteogenomics

Accurate annotation of all protein-coding sequences (CDSs) is an essential prerequisite to fully exploit the rapidly growing repertoire of completely sequenced prokaryotic genomes. However, large discrepancies among the number of CDSs annotated by different resources, missed functional short open reading frames (sORFs), and overprediction of spurious ORFs represent serious limitations.\n\nOur strategy towards accurate and complete genome annotation consolidates CDSs from multiple reference annotation resources, ab initio gene prediction algorithms and in silico ORFs in an integrated proteogenomics database (iPtgxDB) that covers the entire protein-coding potential of a prokaryotic genome. By extending the PeptideClassifier concept of unambiguous peptides for prokaryotes, close to 95% of the identifiable peptides imply one distinct protein, largely simplifying downstream analysis. Searching a comprehensive Bartonella henselae proteomics dataset against such an iPtgxDB allowed us to unambiguously identify novel ORFs uniquely predicted by each resource, including lipoproteins, differentially expressed and membrane-localized proteins, novel start sites and wrongly annotated pseudogenes. Most novelties were confirmed by targeted, parallel reaction monitoring mass spectrometry, including unique ORFs and variants identified in a re-sequenced laboratory strain that are not present in its reference genome. We demonstrate the general applicability of our strategy for genomes with varying GC content and distinct taxonomic origin, and release iPtgxDBs for B. henselae, Bradyrhozibium diazoefficiens and Escherichia coli as well as the software to generate such proteogenomics search databases for any prokaryote.

genomics↗