From crop improvement to breeding healthier livestock to modeling human disease, scientists are using PacBio Sequencing to advance understanding of plant and animal genomes. In this article, we look at four examples of plant and animal genome references improved or made possible with SMRT Sequencing, including an early example of transcriptome sequencing of a chicken for improved annotation. These examples highlight insights gained with SMRT Sequencing that are missed with short-read data, such as complex regions or novel genes.
At the University of Arizona, a leading genomics research facility benefits from decades of BAC- based sequencing expertise, original studies of crop genomes, and a unique emphasis on high molecular weight DNA.
We have isolated several Osiaa23 rice mutants with different knockout genotypes, resulting in different phenotypes, which suggested that different genetic backgrounds or mutation types influence gene function. The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family performs critical roles in auxin signal transduction in plants. In rice, the gene OsIAA23 (Os06t0597000) is known to affect development of roots and shoots, but previous knockouts in OsIAA23 have been sterile and difficult for research continuously. Here, we isolate new Osiaa23 mutants using the CRISPR/Cas9 system in japonica (Wuyunjing24) and indica (Kasalath) rice, with extensive genome re-sequencing to confirm the absence of off-target effects. In Kasalath,…
In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and…
By 2050, there will be 9 billion people on the planet. What will they eat? This is the question that led Rod Wing, Director of the Arizona Genomics Institute, into the field of plant genomics. What has been accomplished so far in the mission to come up with some super green crops? And how does Rod see anti-GMO sentiment and the recent trend toward gluten free diets factoring in? After answering these questions, he dives into a discussion on which sequencing instruments he has used for plant work. Unsurprisingly, Rod prefers the PacBio long reads even though the cost is…
David Kudrna, Rod Wing, and the Arizona Genomics Institute (AGI) plan to fully sequence and annotate the genomes and transcriptomes of 3-4 accessions from each of the estimated 9-15 subpopulation of rice. These subpopulation-specific references will be used to map resequencing data of 3,000 individuals for variation discovery, GWAS, and genomic selection studies to address important traits such as biotic and abiotic stress tolerances, yield, and grain quality. Here Dr. Kudrna presents the first high-quality genome sequence of the rice variety Nagina22. AGI produced and assembled 65-fold coverage of SMRT Sequencing data, resulting in an assembly of 373 Mb with…
Robert Henry, Professor of Innovation in Agriculture and Director of the Queensland Alliance for Agriculture and Food Innovation at the University of Queensland, Australia, discusses how whole genome analysis of Australian wild rice is being used to better understand rice domestication, with the goal of making a diverse genetic resource available for increased rice food security worldwide. The wild “A genome” species represent an effective gene pool for rice. SMRT Sequencing and assembly of two taxon of wild Australian rice has allowed analysis of the relationships with this group. Domesticated rice (Oryza sativa ssp. japonica) nuclear genome shows close relationship…
In a poster presented at AGBT 2017, Fritz Sedlazeck from Johns Hopkins University describes the comparison of genome assemblies produced using long-read PacBio sequencing and short-read sequencing with 10x Genomics scaffolding. An alignment reveals regions missed by the short-read assembly, including repeats, exons, and even whole genes.
At PAG 2017, Rod Wing presented five new, high-quality rice genome assemblies developed with SMRT Sequencing, including one that has eight complete chromosomes including centromeres. He also offered an early look at data generated with the Sequel System for a new assembly underway. This work is done with the goal of developing rice varieties that will be better suited to feeding a rapidly growing global population.
Genomics studies have shown that the insertions, deletions, duplications, translocations, inversions, and tandem repeat expansions in the structural variant (SV) size range (>50 bp) contribute to the evolution of traits and often have significant associations with agronomically important phenotypes. However, most SVs are too small to detect with array comparative genomic hybridization and too large to reliably discover with short-read DNA sequencing. While de novo assembly is the most comprehensive way to identify variants in a genome, recent studies in human genomes show that PacBio SMRT Sequencing sensitively detects structural variants at low coverage. Here we present SV characterization in…
HiFi reads (>99% accurate, 15-20 kb) from the PacBio Sequel II System consistently provide complete and contiguous genome assemblies. In addition to completeness and contiguity, accuracy is of critical importance, as assembly errors complicate downstream analysis, particularly by disrupting gene frames. Metrics used to assess assembly accuracy include: 1) in-frame gene count, 2) kmer consistency, and 3) concordance to a benchmark, where discordances are interpreted as assembly errors. Genome in a Bottle (GIAB) provides a benchmark for the human genome with estimated accuracy of 99.9999% (Q60). Concordance for human HiFi assemblies exceeds Q50, which provides excellent genomes for downstream analysis,…
PacBio’s new Iso-Seq technology allows for rapid generation of full-length cDNA sequences without the need for assembly steps. The technology was tested on leaf mRNA from two model O. sativa ssp. indica cultivars – Minghui 63 and Zhenshan 97. Even though each transcriptome was not exhaustively sequenced, several thousand isoforms described genes over a wide size range, most of which are not present in any currently available FL cDNA collection. In addition, the lack of an assembly requirement provides direct and immediate access to complete mRNA sequences and rapid unraveling of biological novelties.
Since the advent of Next-Generation Sequencing (NGS), the cost of de novo genome sequencing and assembly have dropped precipitately, which has spurred interest in genome sequencing overall. Unfortunately the contiguity of the NGS assembled sequences, as well as the accuracy of these assemblies have suffered. Additionally, most NGS de novo assemblies leave large portions of genomes unresolved, and repetitive regions are often collapsed. When compared to the reference quality genome sequences produced before the NGS era, the new sequences are highly fragmented and often prove to be difficult to properly annotate. In some cases the contiguous portions are smaller than…
Third generation single molecule sequencing technology from Pacific Biosciences, Moleculo, Oxford Nanopore, and other companies are revolutionizing genomics by enabling the sequencing of long, individual molecules of DNA and RNA. One major advantage of these technologies over current short read sequencing is the ability to sequence much longer molecules, thousands or tens of thousands of nucleotides instead of mere hundreds. This capacity gives researchers substantially greater power to probe into microbial, plant, and animal genomes, but it remains unknown on how to best use these data. To answer this, we systematically evaluated the human genome and 25 other important genomes…
Bacterial blight of rice is caused by the ?-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease-susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due…