June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing.

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments generally use short-read, second-generation sequencing, which results in data processing difficulties. For example, reads less than 1 kb in length will likely not cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, single molecule, real-time (SMRT) Sequencing reads in the 1-2 kb range, with >99% accuracy can be efficiently generated for low amounts of input DNA. 10 ng of input DNA sequenced in 4 SMRT Cells would generate >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community, since SMRT Sequencing has been shown to have no sequence-context bias. Long read lengths mean that that it would be reasonable to expect a high number of the reads to include gene fragments useful for analysis.


June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community. Long read lengths translate to a high number of the reads harboring full genes or even full operons for downstream analysis. Here we present the results of circular-consensus sequencing on a mock metagenomic community with an abundance range of multiple orders of magnitude, and compare the results with both 16S and shotgun assembly methods. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows to elucidate meaningful information from the very low-abundance community members. For example, given the above low-input sequencing approach, a community member at 1/1,000 relative abundance would generate 100 1-2 kb sequence fragments having 99% consensus accuracy, with a high probability of containing a gene fragment useful for taxonomic classification or functional insight.


June 1, 2021  |  

A comprehensive lincRNA analysis: From conifers to trees

We have produced an updated annotation of the Norway spruce genome on the basis of an in siliconormalised set of RNA-Seq data obtained from 1,529 samples and comprising 15.5 billion paired-end Illumina HiSeq reads complemented by 18Mbp of PacBio cDNA data (3.2M sequences). In addition to augmenting and refining the previous protein coding gene annotation, here we focus on the addition of long intergenic non-coding RNA (lincRNA) and micro RNA (miRNA) genes. In addition to non-coding loci, our analyses also identified protein coding genes that had been missed by the initial genome annotation and enabled us to update the annotation of existing gene models. In particular, splice variant information, as supported by PacBio sequencing reads, has been added to the current annotation and previously fragmented gene models have been merged by scaffolding disjoint genomic scaffolds on the basis of transcript evidence. Using this refined annotation, a targeted analysis of the lincRNAs enabled their classification as i) deeply conserved, ii) conserved in seed plants iii) gymnosperm/conifer specific. Concurrently, complementary analyses were performed as part of the aspen genome project and the results of a comparative analysis of the lincRNAs conserved in both Norway spruce and Eurasian aspen enabled us to identify conserved and diverged expression profiles. At present, we are delving further into the expression results with the aim to functionally annotate the lincRNA genes, by developing a co-expression network analyses based GO annotation.


June 1, 2021  |  

Profiling the microbiome in fecal microbiota transplantation using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, single molecule, real-time (SMRT®) Sequencing reads in the 1-3kb range, with >99% accuracy can be efficiently generated for low amounts of input DNA. 10 ng of input DNA sequenced in 4 SMRT Cells on the PacBio RS II would generate >100,000 such reads. While throughput is lower compared to short-read sequencing methods, the reads are a true random sampling of the underlying community since SMRT Sequencing has been shown to have very low sequence-context bias. With reads >1 kb at >99% accuracy it is reasonable to expect a high percentage of reads include gene fragments useful for analysis without the need for de novo assembly. Here we present the results of circular consensus sequencing for an individual’s microbiome, before and after undergoing fecal microbiota transplantation (FMT) in order to treat a chronic Clostridium difficile infection. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows us to profile low abundance community members at the species level. We also show that using shotgun sampling with long reads allows a level of functional insight not possible with classic targeted 16S, or short read sequencing, due to entire genes being covered in single reads.


June 1, 2021  |  

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis

Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. The resulting library can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes using the SMRT Portal assembly program HGAP, plus base modification analysis. The entire process can be done in less than 3 days by standard laboratory personnel. This approach is particularly important for analysis of metagenomic communities, in which genomic DNA is often limited. From these samples, full-length 16S amplicons can be generated, prepped with the standard SMRTbell library prep protocol, and sequenced. Alternatively, a 2 kb sheared library, made from a few ng of input DNA, can also be used to elucidate the microbial composition of a community, and may provide information about biochemical pathways present in the sample. In both these cases, 1-2 kb reads with >99.9% accuracy can be obtained from Circular Consensus Sequencing.


June 1, 2021  |  

Workflow for processing high-throughput, Single Molecule, Real-Time Sequencing data for analyzing the microbiome of patients undergoing fecal microbiota transplantation

There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500 bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-3 kb range, with >99% accuracy can be generated using the previous generation PacBio RS II or, in much higher throughput, using the new Sequel System. While throughput is lower compared to short-read sequencing methods, the reads are a true random sampling of the underlying community since SMRT Sequencing has been shown to have very low sequence-context bias. With single-molecule reads >1 kb at >99% consensus accuracy, it is reasonable to expect a high percentage of reads to include genes or gene fragments useful for analysis without the need for de novo assembly. Here we present the results of circular consensus sequencing for an individual’s microbiome, before and after undergoing fecal microbiota transplantation (FMT) in order to treat a chronic Clostridium difficile infection. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows us to profile low abundance community members at the species level. We also show that using shotgun sampling with long reads allows a level of functional insight not possible with classic targeted 16S, or short read sequencing, due to entire genes being covered in single reads.


June 1, 2021  |  

Using the PacBio Sequel System to taxonomically and functionally classify metagenomic samples in a trial of patients undergoing fecal microbiota transplantation

Whole-sample shotgun sequencing can provide a more detailed view of a metagenomic community than 16S sequencing, but its use in multi-sample experiments is limited by throughput, cost and analysis complexity. While short-read sequencing technologies offer higher throughput, read lengthss less fewer than 500 bp will rarely cover a gene of interest, and necessitate assembly before further analysis. Assembling large fragments requires sampling each community member at a high depth, significantly increasing the amount of sequencing needed, and limiting the analysis of rare community members. Assembly methods also risk It is also possible to incorrectly combine combining sequences from different community members.


June 1, 2021  |  

Microbiome profiling at the strain level using rRNA amplicons

Strain level microbiome profiling is needed for a full understanding of how microbial communities influence human health. Microbiome profiling of rRNA gene amplicons is a well-understood method that is rapid and inexpensive, but standard 16S rRNA gene methods generally cannot differentiate closely related strains. Whole genome/shotgun microbiome profiling is considered a higher-resolution alternative, but with decreased throughput and significantly increased sequencing costs and analysis burden. With both methods there are also challenges with microbial lysis, DNA preparation, and taxonomic analysis. Specialized microbiome-focused protocols were developed to achieve strain-level taxonomic differentiation using a rapid, high throughput rRNA gene assay. The protocol integrates lysis and DNA preparation improvements with a unique high information content amplicon and associated novel database to enable taxonomic differentiation of closely related microbial strains.


June 1, 2021  |  

Metagenomic analysis of type II diabetes gut microbiota using PacBio HiFi reads reveals taxonomic and functional differences

In the past decade, the human microbiome has been increasingly shown to play a major role in health. For example, imbalances in gut microbiota appear to be associated with Type II diabetes mellitus (T2DM) and cardiovascular disease. Coronary artery disease (CAD) is a major determinant of the long-term prognosis among T2DM patients, with a 2- to 4-fold increased mortality risk when present. However, the exact microbial strains or functions implicated in disease need further investigation. From a large study with 523 participants (185 healthy controls, 186 T2DM patients without CAD, and 106 T2DM patients with CAD), 3 samples from each patient group were selected for long read sequencing. Each sample was prepared and sequenced on one Sequel II System SMRT Cell, to assess whether long accurate PacBio HiFi reads could yield additional insights to those made using short reads. Each of the 9 samples was subject to metagenomic assembly and binning, taxonomic classification and functional profiling. Results from metagenomic assembly and binning show that it is possible to generate a significant number of complete MAGs (Metagenome Assembled Genomes) from each sample, with over half of the high-quality MAGs being represented by a single circular contig. We show that differences found in taxonomic and functional profiles of healthy versus diabetic patients in the small 9-sample study align with the results of the larger study, as well as with results reported in literature. For example, the abundances of beneficial short- chain fatty acid (SCFA) producers such as Phascolarctobacterium faecium and Faecalibacterium prausnitzii were decreased in T2DM gut microbiota in both studies, while the abundances of quinol and quinone biosynthesis pathways were increased as compared to healthy controls. In conclusion, metagenomic analysis of long accurate HiFi reads revealed important taxonomic and functional differences in T2DM versus healthy gut microbiota. Furthermore, metagenome assembly of long HiFi reads led to the recovery of many complete MAGs and a significant number of complete circular bacterial chromosome sequences.


February 5, 2021  |  

Movie: The new biology

This documentary film features the wave of cutting-edge technologies that now provide the opportunity to create predictive models of living systems, and gain wisdom about the fundamental nature of life…


April 21, 2020  |  

Chryseobacterium mulctrae sp. nov., isolated from raw cow’s milk.

A Gram-stain-negative bacterial strain, designated CA10T, was isolated from bovine raw milk sampled in Anseong, Republic of Korea. Cells were yellow-pigmented, aerobic, non-motile bacilli and grew optimally at 30?°C and pH 7.0 on tryptic soy agar without supplementation of NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain CA10T belonged to the genus Chryseobacterium, family Flavobacteriaceae, and was most closely related to Chryseobacterium indoltheticum ATCC 27950T (98.75?% similarity). The average nucleotide identity and digital DNA-DNA hybridization values of strain CA10T were 94.4 and 56.9?%, respectively, relative to Chryseobacterium scophthalmum DSM 16779T, being lower than the cut-off values of 95-96?and 70?%, respectively. The predominant respiratory quinone was menaquinone-6; major polar lipid, phosphatidylethanolamine; major fatty acids, iso-C15?:?0, summed feature 9 (iso-C17?:?1?9c and/or C16?:?0 10-methyl), summed feature 3 (iso-C15?:?0 2-OH and/or C16?:?1?7c) and iso-C17?:?0 3-OH. The results of physiological, chemotaxonomic and biochemical analyses suggested that strain CA10T is a novel species of genus Chryseobacterium, for which the name Chryseobacterium mulctrae sp. nov. is proposed. The type strain is CA10T (=KACC 21234T=JCM 33443T).


April 21, 2020  |  

Allopseudarcicella aquatilis gen. nov., sp. nov., isolated from freshwater.

A Gram-stain-negative, rod-shaped and red-pigmented strain, HME7025T, was isolated from freshwater sampled in the Republic of Korea. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain HME7025T formed a lineage within the family Cytophagaceae of the phylum Bacteroidetes. Strain HME7025T was closely related to the genera Pseudarcicella, Arcicella and Flectobacillus. The 16S rRNA gene sequence similarity values of strain HME7025T were under 94.5?% to its closest phylogenetic neighbours. The major fatty acids of strain HME7025T were iso-C15?:?0 (41.9?%), summed feature 3 (comprising C16?:?1?7c and/or C16?:?1?6c; 12.2?%) and anteiso-C15?:?0 (10.8?%). The major respiratory quinone was menaquinone-7. The major polar lipids were phosphatidylethanolamine, two unidentified aminophospholipids and one unidentified polar lipid. The DNA G+C content of strain HME7025T was 37.9?mol%. On the basis of the evidence presented in this study, strain HME7025T represents a novel species of a novel genus within the family Cytophagaceae, for which the name Allopseudarcicella aquatilis gen. nov., sp. nov. is proposed. The type strain is HME7025T (=KCTC 23617T=CECT 7957T).


April 21, 2020  |  

The complete genome sequence and comparative genome analysis of the multi-drug resistant food-borne pathogen Bacillus cereus.

Bacillus cereus is an opportunistic human pathogen causing food-borne gastrointestinal infections and non-gastrointestinal infections worldwide. The strain B. cereus FORC_013 was isolated from fried eel. Its genome was completely sequenced by PacBio technology, analyzed and compared with other complete genome sequences of Bacillus to elucidate the distinct pathogenic features of the strain isolated in South Korea. Genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding tissue-destructive exoenzymes, and pore-forming toxins. In particular, tissue-destructive (hemolysin BL, nonhaemolytic enterotoxins) and cytolytic proteins (cytolysin) were observed in the genome, which damage the plasma membrane of the epithelial cells of the small intestine causing diarrhea in humans. Capsule biosynthesis gene found in both chromosome and plasmid, which might be responsible for protecting the pathogen from the host cell immune defense system after host cell invasion. Additionally, multidrug resistance operon and efflux pumps were identified in the genome, which play a prominent role in multi-antibiotic resistance. Comparative phylogenetic tree analysis of the strain FORC_013 and other B. cereus strains revealed that the closest strains are ATCC 14579 and B4264. This genome data can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.Copyright © 2018. Published by Elsevier Inc.


April 21, 2020  |  

Paenibacillus albus sp. nov., a UV radiation-resistant bacterium isolated from soil in Korea.

A novel Gram-stain-positive, motile, white color and endospore-forming bacterium, designated 18JY67-1T, was isolated from soil in Jeju Island, Korea. The strain grow at 15-42 °C (optimum 30 °C) in R2A medium at pH (6.0-9.5) (optimum 7.5). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 18JY67-1T formed a distinct lineage within the family Paenibacillaceae (order Bacillales, class Bacilli), and was closely related to Paenibacillus rhizoryzae (KP675984; 96.9% 16S rRNA gene sequence similarity). The major cellular fatty acids of the strain 18JY67-1T were C16:0 and anteiso-C15:0. The predominant respiratory quinones were MK-7. The major polar lipid was identified as diphosphatidylglycerol. On the basis of phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate 18JY67-1T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus flavus sp. nov. is proposed. The type strain of Paenibacillus flavus is 18JY67-1T (=?KCTC 33959T =?JCM 33184T).


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