June 1, 2021  |  

Getting the most out of your PacBio libraries with size selection.

PacBio RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and demonstrated consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing. The recently launched P5-C3 chemistry generates even longer reads with N50 often >10,000 bp, making it the best choice for scaffolding and spanning structural rearrangements. With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell library itself. Size selection of a high-quality, sheared 20 kb library using the BluePippin™ System has been demonstrated to increase the N50 read length by as much as 5 kb with C3 chemistry. BluePippin size selection or a more stringent AMPure® PB selection cutoff can be used to recover long fragments from degraded genomic material. The selection of chemistries, P4-C2 versus P5-C3, is highly dependent on the final size distribution of the SMRTbell library and experimental goals. PacBio’s long read lengths also allow for the sequencing of full-length cDNA libraries at single-molecule resolution. However, longer transcripts are difficult to detect due to lower abundance, amplification bias, and preferential loading of smaller SMRTbell constructs. Without size selection, most sequenced transcripts are 1-1.5 kb. Size selection dramatically increases the number of transcripts >1.5 kb, and is essential for >3 kb transcripts.

June 1, 2021  |  

Cogent: Reconstructing the coding genome from full-length transcriptome sequences

For highly complex and large genomes, a well-annotated genome may be computationally challenging and costly, yet the study of alternative splicing events and gene annotations usually rely on the existence of a genome. Long-read sequencing technology provides new opportunities to sequence full-length cDNAs, avoiding computational challenges that short read transcript assembly brings. The use of single molecule, real-time sequencing from Pacific Biosciences to sequence transcriptomes (the Iso-SeqTM method), which produces de novo, high-quality, full-length transcripts, has revealed an astonishing amount of alternative splicing in eukaryotic species. With the Iso-Seq method, it is now possible to reconstruct the transcribed regions of the genome using just the transcripts themselves. We present Cogent, a tool for finding gene families and reconstructing the coding genome in the absence of a reference genome. Cogent uses k-mer similarities to first partition the transcripts into different gene families. Then, for each gene family, the transcripts are used to build a splice graph. Cogent identifies bubbles resulting from sequencing errors, minor variants, and exon skipping events, and attempts to resolve each splice graph down to the minimal set of reconstructed contigs. We apply Cogent to a Cuttlefish Iso-Seq dataset, for which there is a highly fragmented, Illumina-based draft genome assembly and little annotation. We show that Cogent successfully discovers gene families and can reconstruct the coding region of gene loci. The reconstructed contigs can then be used to visualize alternative splicing events, identify minor variants, and even be used to improve genome assemblies.

June 1, 2021  |  

Progress Toward a Low Budget Reference Grade Genome Assembly

Reference quality de novo genome assemblies were once solely the domain of large, well-funded genome projects. While next-generation short read technology removed some of the cost barriers, accurate chromosome-scale assembly remains a real challenge. Here we present efforts to de novo assemble the goat (Capra hircus) genome. Through the combination of single-molecule technologies from Pacific Biosciences (sequencing) and BioNano Genomics (optical mapping) coupled with high-throughput chromosome conformation capture sequencing (Hi-C), an inbred San Clemente goat genome has been sequenced and assembled to a high degree of completeness at a relatively modest cost. Starting with 38 million PacBio reads, we integrated the MinHash Alignment Process (MHAP) with the Celera Assembler (CA) to produce an assembly composed of 3110 contigs with a contig N50 size of 4.7 Mb. This assembly was scaffolded with BioNano genome maps derived from a single IrysChip into 333 scaffolds with an N50 of 23.1 Mb including the complete scaffolding of chromosome 20. Finally, cis-chromosome associations were determined by Hi-C, yielding complete reconstruction of all autosomes into single scaffolds with a final N50 of 91.7 Mb. We hope to demonstrate that our methods are not only cost effective, but improve our ability to annotate challenging genomic regions such as highly repetitive immune gene clusters.

June 1, 2021  |  

Best practices for diploid assembly of complex genomes using PacBio: A case study of Cascade Hops

A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over earlier short-read assemblies. The genome is primarily assembled as haplotypes due to the outbred nature of the organism. We explore patterns of haplotype divergence across the assembly and present strategies to deduplicate haplotypes prior to scaffolding

April 9, 2021  |  

Creating Core Demand with HiFi Sequencing

In this video, Dave Miller from PacBio and Alvaro Hernandez PhD from the University of Illinois Urbana- Champaign discuss how to create Core Lab demand using PacBio highly accurate long-read,…

April 21, 2020  |  

Modern technologies and algorithms for scaffolding assembled genomes.

The computational reconstruction of genome sequences from shotgun sequencing data has been greatly simplified by the advent of sequencing technologies that generate long reads. In the case of relatively small genomes (e.g., bacterial or viral), complete genome sequences can frequently be reconstructed computationally without the need for further experiments. However, large and complex genomes, such as those of most animals and plants, continue to pose significant challenges. In such genomes, assembly software produces incomplete and fragmented reconstructions that require additional experimentally derived information and manual intervention in order to reconstruct individual chromosome arms. Recent technologies originally designed to capture chromatin structure have been shown to effectively complement sequencing data, leading to much more contiguous reconstructions of genomes than previously possible. Here, we survey these technologies and the algorithms used to assemble and analyze large eukaryotic genomes, placed within the historical context of genome scaffolding technologies that have been in existence since the dawn of the genomic era.

April 21, 2020  |  

Sequencing of Cultivated Peanut, Arachis hypogaea, Yields Insights into Genome Evolution and Oil Improvement.

Cultivated peanut (Arachis hypogaea) is an allotetraploid crop planted in Asia, Africa, and America for edible oil and protein. To explore the origins and consequences of tetraploidy, we sequenced the allotetraploid A. hypogaea genome and compared it with the related diploid Arachis duranensis and Arachis ipaensis genomes. We annotated 39 888 A-subgenome genes and 41 526 B-subgenome genes in allotetraploid peanut. The A. hypogaea subgenomes have evolved asymmetrically, with the B subgenome resembling the ancestral state and the A subgenome undergoing more gene disruption, loss, conversion, and transposable element proliferation, and having reduced gene expression during seed development despite lacking genome-wide expression dominance. Genomic and transcriptomic analyses identified more than 2 500 oil metabolism-related genes and revealed that most of them show altered expression early in seed development while their expression ceases during desiccation, presenting a comprehensive map of peanut lipid biosynthesis. The availability of these genomic resources will facilitate a better understanding of the complex genome architecture, agronomically and economically important genes, and genetic improvement of peanut.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

April 21, 2020  |  

Inter-chromosomal coupling between vision and pigmentation genes during genomic divergence.

Recombination between loci underlying mate choice and ecological traits is a major evolutionary force acting against speciation with gene flow. The evolution of linkage disequilibrium between such loci is therefore a fundamental step in the origin of species. Here, we show that this process can take place in the absence of physical linkage in hamlets-a group of closely related reef fishes from the wider Caribbean that differ essentially in colour pattern and are reproductively isolated through strong visually-based assortative mating. Using full-genome analysis, we identify four narrow genomic intervals that are consistently differentiated among sympatric species in a backdrop of extremely low genomic divergence. These four intervals include genes involved in pigmentation (sox10), axial patterning (hoxc13a), photoreceptor development (casz1) and visual sensitivity (SWS and LWS opsins) that develop islands of long-distance and inter-chromosomal linkage disequilibrium as species diverge. The relatively simple genomic architecture of species differences facilitates the evolution of linkage disequilibrium in the presence of gene flow.

September 22, 2019  |  

L_RNA_scaffolder: scaffolding genomes with transcripts.

Generation of large mate-pair libraries is necessary for de novo genome assembly but the procedure is complex and time-consuming. Furthermore, in some complex genomes, it is hard to increase the N50 length even with large mate-pair libraries, which leads to low transcript coverage. Thus, it is necessary to develop other simple scaffolding approaches, to at least solve the elongation of transcribed fragments.We describe L_RNA_scaffolder, a novel genome scaffolding method that uses long transcriptome reads to order, orient and combine genomic fragments into larger sequences. To demonstrate the accuracy of the method, the zebrafish genome was scaffolded. With expanded human transcriptome data, the N50 of human genome was doubled and L_RNA_scaffolder out-performed most scaffolding results by existing scaffolders which employ mate-pair libraries. In these two examples, the transcript coverage was almost complete, especially for long transcripts. We applied L_RNA_scaffolder to the highly polymorphic pearl oyster draft genome and the gene model length significantly increased.The simplicity and high-throughput of RNA-seq data makes this approach suitable for genome scaffolding. L_RNA_scaffolder is available at http://www.fishbrowser.org/software/L_RNA_scaffolder.

September 22, 2019  |  

Lentinula edodes genome survey and postharvest transcriptome analysis.

Lentinula edodes is a popular, cultivated edible and medicinal mushroom. Lentinula edodes is susceptible to postharvest problems, such as gill browning, fruiting body softening, and lentinan degradation. We constructed a de novo assembly draft genome sequence and performed gene prediction for Lentinula edodesDe novo assembly was carried out using short reads from paired-end and mate-paired libraries and by using long reads by PacBio, resulting in a contig number of 1,951 and an N50 of 1 Mb. Furthermore, we predicted genes by Augustus using transcriptome sequencing (RNA-seq) data from the whole life cycle of Lentinula edodes, resulting in 12,959 predicted genes. This analysis revealed that Lentinula edodes lacks lignin peroxidase. To reveal genes involved in the loss of quality of Lentinula edodes postharvest fruiting bodies, transcriptome analysis was carried out using serial analysis of gene expression (SuperSAGE). This analysis revealed that many cell wall-related enzymes are upregulated after harvest, such as ß-1,3-1,6-glucan-degrading enzymes in glycoside hydrolase (GH) families GH5, GH16, GH30, GH55, and GH128, and thaumatin-like proteins. In addition, we found that several chitin-related genes are upregulated, such as putative chitinases in GH family 18, exochitinases in GH20, and a putative chitosanase in GH family 75. The results suggest that cell wall-degrading enzymes synergistically cooperate for rapid fruiting body autolysis. Many putative transcription factor genes were upregulated postharvest, such as genes containing high-mobility-group (HMG) domains and zinc finger domains. Several cell death-related proteins were also upregulated postharvest.IMPORTANCE Our data collectively suggest that there is a rapid fruiting body autolysis system in Lentinula edodes The genes for the loss of postharvest quality newly found in this research will be targets for the future breeding of strains that keep fresh longer than present strains. De novoLentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding. Copyright © 2017 American Society for Microbiology.

September 22, 2019  |  

Single-cell (meta-)genomics of a dimorphic Candidatus Thiomargarita nelsonii reveals genomic plasticity.

The genus Thiomargarita includes the world’s largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita’s core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.

September 22, 2019  |  

Sequence of the sugar pine megagenome.

Until very recently, complete characterization of the megagenomes of conifers has remained elusive. The diploid genome of sugar pine (Pinus lambertiana Dougl.) has a highly repetitive, 31 billion bp genome. It is the largest genome sequenced and assembled to date, and the first from the subgenus Strobus, or white pines, a group that is notable for having the largest genomes among the pines. The genome represents a unique opportunity to investigate genome “obesity” in conifers and white pines. Comparative analysis of P. lambertiana and P. taeda L. reveals new insights on the conservation, age, and diversity of the highly abundant transposable elements, the primary factor determining genome size. Like most North American white pines, the principal pathogen of P. lambertiana is white pine blister rust (Cronartium ribicola J.C. Fischer ex Raben.). Identification of candidate genes for resistance to this pathogen is of great ecological importance. The genome sequence afforded us the opportunity to make substantial progress on locating the major dominant gene for simple resistance hypersensitive response, Cr1 We describe new markers and gene annotation that are both tightly linked to Cr1 in a mapping population, and associated with Cr1 in unrelated sugar pine individuals sampled throughout the species’ range, creating a solid foundation for future mapping. This genomic variation and annotated candidate genes characterized in our study of the Cr1 region are resources for future marker-assisted breeding efforts as well as for investigations of fundamental mechanisms of invasive disease and evolutionary response. Copyright © 2016 by the Genetics Society of America.

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