June 1, 2021  |  

Genome sequencing of endosymbiotic bacterial Streptomyces sp. from Antartic lichen using Single Molecule Real-time Sequencing (SMRT) technology.

Along with the advent of next-generation sequencing (NGS) techniques, it has become possible to sequence a microbial genome very quickly with high coverage. Recently, PacificBioscience developed single molecule real-time sequencing (SMRT) technology, 3rd generation sequencing platform, which provide much longer (average read length: 1.5Kb) reads without PCR amplification. We did de novo sequencing of Streptomyces sp. using Illumina GAIIx, Roche 454 and PacBio RS system and compared the data. The endosymbiotic bacteria Streptomyces sp. PAMC 26508 was isolated from Antarctic lichen Psoroma sp. that grows attached rocks on Barton Peninsula, King George Island, Antarctica (62, 13’S, 58, 47’W). With 4 SMRT cells, we could get more than 15x coverage of corrected sequence data for de novo assembly. Comparing the performance of other sequencing platforms, PacBio platform could generate data on similar manner with general mid-level GC content organism. In conclusion, PacBio RS system, SMRT technology, shows better performance with high GC content organisms and is expected to be the new tool to improve the de novo sequencing and assembly.


June 1, 2021  |  

SMRT Sequencing solutions for large genomes and transcriptomes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers in large genome complexities, such as long, highly repetitive, low-complexity regions and duplication events, and differentiating between transcript isoforms that are difficult to resolve with short-read technologies. We present solutions available for both reference genome improvement (>100 MB) and transcriptome research to best leverage long reads that have exceeded 20 Kb in length. Benefits for these applications are further realized with consistent use of size-selection of input sample using the BluePippin™ device from Sage Science. Highlights from our genome assembly projects using the latest P5-C3 chemistry on model organisms will be shared. Assembly contig N50 have exceeded 6 Mb and we observed longest contig exceeding 12.5 Mb with an average base quality of QV50. Additionally, the value of long, intact reads to provide a no-assembly approach to investigate transcript isoforms using our Iso-Seq Application will be presented.


June 1, 2021  |  

A comparison of assemblers and strategies for complex, large-genome sequencing with PacBio long reads.

PacBio sequencing holds promise for addressing large-genome complexities, such as long, highly repetitive, low-complexity regions and duplication events that are difficult to resolve with short-read technologies. Several strategies, with varying outcomes, are available for de novo sequencing and assembling of larger genomes. Using a diploid fungal genome, estimated to be ~80 Mb in size, as the basis dataset for comparison, we highlight assembly options when using only PacBio sequencing or a combined strategy leveraging data sets from multiple sequencing technologies. Data generated from SMRT Sequencing was subjected to assembly using different large-genome assemblers, and comparisons of the results will be shown. These include results generated with HGAP, Celera Assembler, MIRA, PBJelly, and other assembly tools currently in development. Improvements observed include a near 50% reduction in the number of contigs coupled with at least a doubling of contig N50 size in genome assemblies incorporating SMRT Sequencing data. We further show how incorporating long reads also highlights new challenges and missed insights of short-read assemblies arising from heterozygosity inherent in multiploid genomes.


June 1, 2021  |  

Best practices for whole-genome de novo sequencing with long-read SMRT Sequencing.

With the introduction of P6-C4 chemistry, PacBio has made significant strides with Single Molecule, Real-Time (SMRT) Sequencing . Read lengths averaging between 10 and 15 kb can be now be achieved with extreme reads in the distribution of > 60 kb. The chemistry attains a consensus accuracy of 99.999% (QV50) at 30x coverage which coupled with an increased throughput from the PacBio RS II platform (500 Mb – 1 Gb per SMRT Cell) makes larger genome projects more tractable. These combined advancements in technology deliver results that rival the quality of Sanger “clone-by-clone” sequencing efforts; resulting in closed microbial genomes and highly contiguous de novo assembly of complex eukaryotes on multi-Gbase scale using SMRT Sequencing as the standalone technology. We present here the guidelines and best practices to achieve optimal results when employing PacBio-only whole genome shotgun sequencing strategies. Specific sequencing examples for plant and animal genomes are discussed with SMRTbell library preparation and purification methods for obtaining long insert libraries to generate optimal sequencing results. The benefits of long reads are demonstrated by the highly contiguous assemblies yielding contig N50s of over 5 Mb compared to similar assemblies using next-generation short-read approaches. Finally, guidelines will be presented for planning out projects for the de novo assembly of large genomes.


April 21, 2020  |  

Lateral transfers of large DNA fragments spread functional genes among grasses.

A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism’s adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grass Alloteropsis semialata, a species surmised to exhibit two LGTs, and screen it for other grass-to-grass LGTs using genomic data from 146 other grass species. Through stringent phylogenomic analyses, we discovered 57 additional LGTs in the A. semialata nuclear genome, involving at least nine different donor species. The LGTs are clustered in 23 laterally acquired genomic fragments that are up to 170 kb long and have accumulated during the diversification of Alloteropsis. The majority of the 59 LGTs in A. semialata are expressed, and we show that they have added functions to the recipient genome. Functional LGTs were further detected in the genomes of five other grass species, demonstrating that this process is likely widespread in this globally important group of plants. LGT therefore appears to represent a potent evolutionary force capable of spreading functional genes among distantly related grass species. Copyright © 2019 the Author(s). Published by PNAS.


April 21, 2020  |  

A comprehensive evaluation of long read error correction methods

Motivation: Third-generation sequencing technologies can sequence long reads, which is advancing the frontiers of genomics research. However, their high error rates prohibit accurate and efficient downstream analysis. This difficulty has motivated the development of many long read error correction tools, which tackle this problem through sampling redundancy and/or leveraging accurate short reads of the same biological samples. Existing studies to asses these tools use simulated data sets, and are not sufficiently comprehensive in the range of software covered or diversity of evaluation measures used. Results: In this paper, we present a categorization and review of long read error correction methods, and provide a comprehensive evaluation of the corresponding long read error correction tools. Leveraging recent real sequencing data, we establish benchmark data sets and set up evaluation criteria for a comparative assessment which includes quality of error correction as well as run-time and memory usage. We study how trimming and long read sequencing depth affect error correction in terms of length distribution and genome coverage post-correction, and the impact of error correction performance on an important application of long reads, genome assembly. We provide guidelines for practitioners for choosing among the available error correction tools and identify directions for future research.


April 21, 2020  |  

Survey of the Bradysia odoriphaga Transcriptome Using PacBio Single-Molecule Long-Read Sequencing.

The damage caused by Bradysia odoriphaga is the main factor threatening the production of vegetables in the Liliaceae family. However, few genetic studies of B. odoriphaga have been conducted because of a lack of genomic resources. Many long-read sequencing technologies have been developed in the last decade; therefore, in this study, the transcriptome including all development stages of B. odoriphaga was sequenced for the first time by Pacific single-molecule long-read sequencing. Here, 39,129 isoforms were generated, and 35,645 were found to have annotation results when checked against sequences available in different databases. Overall, 18,473 isoforms were distributed in 25 various Clusters of Orthologous Groups, and 11,880 isoforms were categorized into 60 functional groups that belonged to the three main Gene Ontology classifications. Moreover, 30,610 isoforms were assigned into 44 functional categories belonging to six main Kyoto Encyclopedia of Genes and Genomes functional categories. Coding DNA sequence (CDS) prediction showed that 36,419 out of 39,129 isoforms were predicted to have CDS, and 4319 simple sequence repeats were detected in total. Finally, 266 insecticide resistance and metabolism-related isoforms were identified as candidate genes for further investigation of insecticide resistance and metabolism in B. odoriphaga.


April 21, 2020  |  

Tools and Strategies for Long-Read Sequencing and De Novo Assembly of Plant Genomes.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

De novo assembly of white poplar genome and genetic diversity of white poplar population in Irtysh River basin in China.

The white poplar (Populus alba) is widely distributed in Central Asia and Europe. There are natural populations of white poplar in Irtysh River basin in China. It also can be cultivated and grown well in northern China. In this study, we sequenced the genome of P. alba by single-molecule real-time technology. De novo assembly of P. alba had a genome size of 415.99 Mb with a contig N50 of 1.18 Mb. A total of 32,963 protein-coding genes were identified. 45.16% of the genome was annotated as repetitive elements. Genome evolution analysis revealed that divergence between P. alba and Populus trichocarpa (black cottonwood) occurred ~5.0 Mya (3.0, 7.1). Fourfold synonymous third-codon transversion (4DTV) and synonymous substitution rate (ks) distributions supported the occurrence of the salicoid WGD event (~ 65 Mya). Twelve natural populations of P. alba in the Irtysh River basin in China were sequenced to explore the genetic diversity. Average pooled heterozygosity value of P. alba populations was 0.170±0.014, which was lower than that in Italy (0.271±0.051) and Hungary (0.264±0.054). Tajima’s D values showed a negative distribution, which might signify an excess of low frequency polymorphisms and a bottleneck with later expansion of P. alba populations examined.


April 21, 2020  |  

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes. Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.


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