June 1, 2021  |  

Single Molecule, Real-Time Sequencing for base modification detection in eukaryotic organisms: Coprinopsis cinerea.

Single Molecule Real-Time (SMRT) DNA sequencing provides a wealth of kinetic information beyond the extraction of the primary DNA sequence, and this kinetic information can provide for the direct detection of modified bases present in genomic DNA. This method has been demonstrated for base modification detection in prokaryotes at base and strand resolutions. In eukaryotes, the common base modifications known to exist are the cytosine variants including methyl, hydroxymethyl, formyl and carboxyl forms. Each of these modifications exhibits different signatures in SMRT kinetic data, allowing for unprecedented possibilities to differentiate between them in direct sequencing data. We present early results of directly sequencing different base modifications in eukaryotic genomic DNA using this method.


June 1, 2021  |  

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark discoveries in biochemistry and genetics. We show that de novo assembly of a new strain yields complete assemblies of entire chromosomes, and additionally contains entire centromeric sequences. Base-modification analyses reveal candidate sites of increased interpulse duration (IPD) ratio, that may signify regions of 5mC, 5hmC, or 6mA base modifications. Iso-seq method provides full-length transcript evidence for comprehensive gene annotation, as well as context to the base-modifications in the newly assembled genome. Projects that integrate multiple genome-wide assays could become common practice for identifying genomic elements and understanding their function in new strains and organisms.


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  |  

The resurgence of reference quality genome sequence.

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 the average gene size making the sequence not nearly as useful for biologists as the earlier reference quality genomes including of Human, Mouse, C. elegans, or Drosophila. Recently, new 3rd generation sequencing technologies, long-range molecular techniques, and new informatics tools have facilitated a return to high quality assembly. We will discuss the capabilities of the technologies and assess their impact on assembly projects across the tree of life from small microbial and fungal genomes through large plant and animal genomes. Beyond improvements to contiguity, we will focus on the additional biological insights that can be made with better assemblies, including more complete analysis genes in their flanking regulatory context, in-depth studies of transposable elements and other complex gene families, and long-range synteny analysis of entire chromosomes. We will also discuss the need for new algorithms for representing and analyzing collections of many complete genomes at once.


June 1, 2021  |  

The Wild Vigna

PacBio 2015 User Group Meeting Presentation Slides: Ken Naito of the NIAS Genetic Resource Center presented on whole genome sequencing of the azuki bean (Vigna angularis). Using single molecule real-time (SMRT) sequencing technology, they achieved the best contiguity and coverage among currently assembled legume crops.


June 1, 2021  |  

Diploid genome assembly and comprehensive haplotype sequence reconstruction

Outside of the simplest cases (haploid, bacteria, or inbreds), genomic information is not carried in a single reference per individual, but rather has higher ploidy (n=>2) for almost all organisms. The existence of two or more highly related sequences within an individual makes it extremely difficult to build high quality, highly contiguous genome assemblies from short DNA fragments. Based on the earlier work on a polyploidy aware assembler, FALCON ( https://github.com/PacificBiosciences/FALCON) , we developed new algorithms and software (“FALCON-unzip”) for de novo haplotype reconstructions from SMRT Sequencing data. We generate two datasets for developing the algorithms and the prototype software: (1) whole genome sequencing data from a highly repetitive diploid fungal (Clavicorona pyxidata) and (2) whole genome sequencing data from an F1 hybrid from two inbred Arabidopsis strains: Cvi-0 and Col-0. For the fungal genome, we achieved an N50 of 1.53 Mb (of the 1n assembly contigs) of the ~42 Mb 1n genome and an N50 of the haplotigs (haplotype specific contigs) of 872 kb from a 95X read length N50 ~16 kb dataset. We found that ~ 45% of the genome was highly heterozygous and ~55% of the genome was highly homozygous. We developed methods to assess the base-level accuracy and local haplotype phasing accuracy of the assembly with short-read data from the Illumina® platform. For the ArabidopsisF1 hybrid genome, we found that 80% of the genome could be separated into haplotigs. The long range accuracy of phasing haplotigs was evaluated by comparing them to the assemblies from the two inbred parental lines. We show that a more complete view of all haplotypes could provide useful biological insights through improved annotation, characterization of heterozygous variants of all sizes, and resolution of differential allele expression. The current Falcon-Unzip method will lead to understand how to solve more difficult polyploid genome assembly problems and improve the computational efficiency for large genome assemblies. Based on this work, we can develop a pipeline enabling routinely assemble diploid or polyploid genomes as haplotigs, representing a comprehensive view of the genomes that can be studied with the information at hand.


June 1, 2021  |  

Phased diploid genome assembly with single-molecule real-time sequencing

While genome assembly projects have been successful in many haploid and inbred species, the assembly of non-inbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short- or long-read approaches. The phased diploid assembly enabled the study of haplotype structure and heterozygosities between homologous chromosomes, including the identification of widespread heterozygous structural variation within coding sequences.


June 1, 2021  |  

Applying Sequel to Genomic Datasets

De novo assembly is a large part of JGI’s analysis portfolio. Repetitive DNA sequences are abundant in a wide range of organisms we sequence and pose a significant technical challenge for assembly. We are interested in long read technologies capable of spanning genomic repeats to produce better assemblies. We currently have three RS II and two Sequel PacBio machines. RS II machines are primarily used for fungal and microbial genome assembly as well as synthetic biology validation. Between microbes and fungi we produce hundreds of PacBio libraries a year and for throughput reasons the vast majority of these are >10 kb AMPure libraries. Throughput for RS II is about 1 Gb per SMRT Cell. This is ideal for microbial sized genomes but can be costly and labor intensive for larger projects which require multiple cells. JGI was an early access site for Sequel and began testing with real samples in January 2016. During that time we’ve had the opportunity to sequence microbes, fungi, metagenomes, and plants. Here we present our experience over the last 18 months using the Sequel platform and provide comparisons with RS II results.


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