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June 1, 2021  |  

Resolving the ‘dark matter’ in genomes.

Second-generation sequencing has brought about tremendous insights into the genetic underpinnings of biology. However, there are many functionally important and medically relevant regions of genomes that are currently difficult or impossible to sequence, resulting in incomplete and fragmented views of genomes. Two main causes are (i) limitations to read DNA of extreme sequence content (GC-rich or AT-rich regions, low complexity sequence contexts) and (ii) insufficient read lengths which leave various forms of structural variation unresolved and result in mapping ambiguities.


June 1, 2021  |  

The use of PacBio and Hi-C data in de novo assembly of the goat genome.

Generating de novo reference genome assemblies for non-model organisms is a laborious task that often requires a large amount of data from several sequencing platforms and cytogenetic surveys. By using PacBio sequence data and new library creation techniques, we present a de novo, high quality reference assembly for the goat (Capra hircus) that demonstrates a primarily sequencing-based approach to efficiently create new reference assemblies for Eukaryotic species. This goat reference genome was created using 38 million PacBio P5-C3 reads generated from a San Clemente goat using the Celera Assembler PBcR pipeline with PacBio read self-correction. In order to generate the assembly, corrected and filtered reads were pre-assembled into a consensus model using PBDAGCON, and subsequently assembled using the Celera Assembly version 8.2. We generated 5,902 contigs using this method with a contig N50 size of 2.56 megabases. In order to generate chromosome-sized scaffolds, we used the LACHESIS scaffolding method to identify cis-chromosome Hi-C interactions in order to link contigs together. We then compared our new assembly to the existing goat reference assembly to identify large-scale discrepancies. In our comparison, we identified 247 disagreements between the two assemblies consisting of 123 inversions and 124 chromosome-contig relocations. The high quality of this data illustrates how this methodology can be used to efficiently generate new reference genome assemblies without the use of expensive fluorescent cytometry or large quantities of data from multiple sequencing platforms.


June 1, 2021  |  

Progress on the reassembly and annotation of the goat genome.

The goat (Capra hircus) remains an important livestock species due to the species’ ability to forage and provide milk, meat and wool in arid environments. The current goat reference assembly and annotation borrows heavily from other loosely related livestock species, such as cattle, and may not reflect the unique structural and functional characteristics of the species. We present preliminary data from a new de novo reference assembly for goat that primarily utilizes 38 million PacBio P5-C3 reads generated from an inbred San Clemente goat. This assembly consists of only 5,902 contigs with a contig N50 size of 2.56 megabases which were grouped into scaffolds using cis-chromosome associations generated by the analysis of Hi-C sequence reads. To provide accurate functional genetic annotation, we utilized existing RNA-seq data and generated new data consisting of over 784 million reads from a combination of 27 different developmental timepoints/tissues. This dataset provides a tangible improvement over existing goat genomics resources by correcting over 247 misassemblies in the current goat reference genome and by annotating predicted gene models with actual expressed transcript data. Our goal is to provide a high quality resource to researchers to enable future genomic selection and functional prediction within the field of goat genomics.


June 1, 2021  |  

Draft genome of horseweed illuminates expansion of gene families that might endow herbicide resistance.

Conyza canadensis (horseweed), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n=2X=18) with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic basis of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000 and PacBio RS) using various libraries with different insertion sizes (~350 bp, ~600 bp, ~3 kb and ~10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (~350× coverage) of data, the genome was assembled into 13,966 scaffolds with N50 =33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (~153 kb) and a nearly-complete mitochondrial genome (~450 kb in 120 scaffolds). The nuclear genome is comprised of 44,592 protein-coding genes. Genome re-sequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or –susceptible biotypes. The draft genome will be useful to better understand weediness, the evolution of herbicide resistance, and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this paper represents the first published draft genome of an agricultural weed.


June 1, 2021  |  

Old school/new school genome sequencing: One step backward — a quantum leap forward.

As the costs for genome sequencing have decreased the number of “genome” sequences have increased at a rapid pace. Unfortunately, the quality and completeness of these so–called “genome” sequences have suffered enormously. We prefer to call such genome assemblies as “gene assembly space” (GAS). We believe it is important to distinguish GAS assemblies from reference genome assemblies (RGAs) as all subsequent research that depends on accurate genome assemblies can be highly compromised if the only assembly available is a GAS assembly.


June 1, 2021  |  

A genome assembly of the domestic goat from 70x coverage of single molecule, real-time sequence.

Goat is an important source of milk, meat, and fiber, especially in developing countries. An advantage of goats as livestock is the low maintenance requirements and high adaptability compared to other milk producers. The global population of domestic goats exceeds 800 million. In Africa, goat production is characterized by low productivity levels, and attempts to introduce more productive breeds have met with poor success due in part to nutritional constraints. It has been suggested that incorporation of selective breeding within the herds adapted for survival could represent one approach to improving food security across Africa. A recently produced genome assembly of a Chinese Yunnan breed goat, based on 192 Gb of short reads across a range of insert sizes from 180 bp to 20 kb, reported a contig N50 of 18.7 kb. The scaffold N50 was improved from 2.2 Mb to 3.1 Mb by addition of fosmid end sequence, with an estimated 140 million Ns in gaps and 91% coverage. The assembly has proven somewhat problematic for pursuing genome-wide association analysis with SNP arrays, apparently due in part to errors in ordering of markers using the draft genome. In order to provide a higher quality assembly, we sequenced a highly inbred, San Clemente breed goat genome using 458 SMRT cells on the Pacific Biosciences platform. These cells generated 193.5 Gbases of sequence after processing into subreads, with mean 5110 bases and max subread length of 40.5 kb. This sequence data generated an assembly using the recently reported MHAP error correction approach and Celera Assembler v8.2. The contig N50 was 2.5 Mb, with the largest contig spanning 19.5 Mb. Additional characteristics of the assembly will be presented.


June 1, 2021  |  

De novo assembly of a complex panicoid grass genome using ultra-long PacBio reads with P6C4 chemistry

Drought is responsible for much of the global losses in crop yields and understanding how plants naturally cope with drought stress is essential for breeding and engineering crops for the changing climate. Resurrection plants desiccate to complete dryness during times of drought, then “come back to life” once water is available making them an excellent model for studying drought tolerance. Understanding the molecular networks governing how resurrection plants handle desiccation will provide targets for crop engineering. Oropetium thomaeum (Oro) is a resurrection plant that also has the smallest known grass genome at 250 Mb compared to Brachypodium distachyon (300 Mb) and rice (350 Mb). Plant genomes, especially grasses, have complex repeat structures such as telomeres, centromeres, and ribosomal gene cassettes, and high heterozygosity, which makes them difficult to assembly using short read next generation sequencing technologies. Ultra-long PacBio reads using the new P6C4 chemistry and the latest 15kb Blue Pippin size-selection protocol to generate 20kb insert libraries that yielded an average read length of 12kb providing ~72X coverage, and 10X coverage with reads over 20kb. The HGAP assembly covers 98% of the genome with a contig N50 of 2.4 Mb, which makes it one of the highest quality and most complete plant genomes assembled to date. Oro has a compact genome structure compared to other grasses with only 16% repeat sequences but has very good collinearity with other grasses. Understanding the genomic mechanisms of extreme desiccation tolerance in resurrection plants like Oro will provide insights for engineering and intelligent breeding of improved food, fuel, and fiber crops.


June 1, 2021  |  

Genome assembly strategies of the recent polyploid, Coffea arabica.

Arabica coffee, revered for its taste and aroma, has a complex genome. It is an allotetraploid (2n=4x=44) with a genome size of approximately 1.3 Gb, derived from the recent (< 0.6 Mya) hybridization of two diploid progenitors (2n=2x=22), C. canephora (710 Mb) and C. eugenioides (670 Mb). Both parental species diverged recently (< 4.2Mya) and their genomes are highly homologous. To facilitate assembly, a dihaploid plant was chosen for sequencing. Initial genome assembly attempts with short read data produced an assembly covering 1,031 Mb of the C. arabica genome with a contig L50 of 9kb. By implementation of long read PacBio at greater than 50x coverage and cutting-edge PacBio software, a de novo PacBio-only genome assembly was constructed that covers 1,042 Mb of the genome with an L50 of 267 kb. The two assemblies were assessed and compared to determine gene content, chimeric regions, and the ability to separate the parental genomes. A genetic map that contains 600 SSRs is being used for anchoring the contigs and improve the sub-genome differentiation together with the search of sub-genome specific SNPs. PacBio transcriptome sequencing is currently being added to finalize gene annotation of the polished assembly. The finished genome assembly will be used to guide re-sequencing assemblies of parental genomes (C. canephora and C. eugenioides) as well as a template for GBS analysis and whole genome re-sequencing of a set of C. arabica accessions representative of the species diversity. The obtained data will provide powerful genomic tools to enable more efficient coffee breeding strategies for this crop, which is highly susceptible to climate change and is the main source of income for millions of small farmers in producing countries.


June 1, 2021  |  

Resources for advanced bioinformaticians working in plant and animal genomes with SMRT Sequencing.

Significant advances in bioinformatics tool development have been made to more efficiently leverage and deliver high-quality genome assemblies with PacBio long-read data. Current data throughput of SMRT Sequencing delivers average read lengths ranging from 10-15 kb with the longest reads exceeding 40 kb. This has resulted in consistent demonstration of a minimum 10-fold improvement in genome assemblies with contig N50 in the megabase range compared to assemblies generated using only short- read technologies. This poster highlights recent advances and resources available for advanced bioinformaticians and developers interested in the current state-of-the-art large genome solutions available as open-source code from PacBio and third-party solutions, including HGAP, MHAP, and ECTools. Resources and tools available on GitHub are reviewed, as well as datasets representing major model research organisms made publically available for community evaluation or interested developers.


June 1, 2021  |  

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.


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  |  

Toward comprehensive genomics analysis with de novo assembly.

Whole genome sequencing can provide comprehensive information important for determining the biochemical and genetic nature of all elements inside a genome. The high-quality genome references produced from past genome projects and advances in short-read sequencing technologies have enabled quick and cheap analysis for simple variants. However even with the focus on genome-wide resequencing for SNPs, the heritability of more than 50% of human diseases remains elusive. For non-human organisms, high-contiguity references are deficient, limiting the analysis of genomic features. The long and unbiased reads from single molecule, real-time (SMRT) Sequencing and new de novo assembly approaches have demonstrated the ability to detect more complicated variants and chromosome-level phasing. Moreover, with the recent advance of bioinformatics algorithms and tools, the computation tasks for completing high-quality de novo assembly of large genomes becomes feasible with commodity hardware. Ongoing development in sequencing technologies and bioinformatics will likely lead to routine generation of high-quality reference assemblies in the future. We discuss the current state of art and the challenges in bioinformatics toward such a goal. More specifically, explicit examples of pragmatic computational requirements for assembling mammalian-size genomes and algorithms suitable for processing diploid genomes are discussed.


June 1, 2021  |  

Whole genome sequencing and epigenome characterization of cancer cells using the PacBio platform.

The comprehensive characterization of cancer genomes and epigenomes for understanding drug resistance remains an important challenge in the field of oncology. For example, PC-9, a non-small cell lung cancer (NSCL) cell line, contains a deletion mutation in exon 19 (DelE746A750) of EGRF that renders it sensitive to erlotinib, an EGFR inhibitor. However, sustained treatment of these cells with erlotinib leads to drug-tolerant cell populations that grow in the presence of erlotinib. However, the resistant cells can be resensitized to erlotinib upon treatment with methyltransferase inhibitors, suggesting a role of epigenetic modification in development of drug resistance. We have characterized for the first time cancer genomes of both drug-sensitive and drug-resistant PC- 9 cells using long-read PacBio sequencing. The PacBio data allowed us to generate a high-quality, de novo assembly of this cancer genome, enabling the detection of forms of genomic variations at all size scales, including SNPs, structural variations, copy number alterations, gene fusions, and translocations. The data simultaneously provide a global view of epigenetic DNA modifications such as methylation. We will present findings on large-scale changes in the methylation status across the cancer genome as a function of drug sensitivity.


June 1, 2021  |  

Assembly of complete KIR haplotypes from a diploid individual by the direct sequencing of full-length fosmids.

We show that linearizing and directly sequencing full-length fosmids simplifies the assembly problem such that it is possible to unambiguously assemble individual haplotypes for the highly repetitive 100-200 kb killer Ig-like receptor (KIR) gene loci of chromosome 19. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the KIR locus, means that sequence assembly of pooled samples into complete haplotypes is difficult and in many cases impossible. The current maximum read length generated by SMRT Sequencing exceeds the length of a 40 kb fosmid; it is therefore possible to span an entire fosmid in one sequencing read. Shearing, sequencing and assembling fosmids in a shotgun approach is prone to errors when the underlying sequence is highly repetitive. We show that it is possible to directly sequence linearized fosmids and generate a high-quality consensus by simple alignment, removing the need for an error-prone assembly step. The high-quality sequence of complete fosmids can then be tiled into full haplotypes. We demonstrate the method on DNA samples from a number of individuals and fully recover the sequence of both haplotypes from a pool of KIR fosmids. The ability to haplotype and sequence complex immunogenetic regions will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. This simple and robust approach can be scaled-up allowing a complex genomic region to be sequenced at a population level. We expect such sequencing to be valuable in disease association research.


June 1, 2021  |  

Highly contiguous de novo human genome assembly and long-range haplotype phasing using SMRT Sequencing

The long reads, random error, and unbiased sampling of SMRT Sequencing enables high quality, de novo assembly of the human genome. PacBio long reads are capable of resolving genomic variations at all size scales, including SNPs, insertions, deletions, inversions, translocations, and repeat expansions, all of which are important in understanding the genetic basis for human disease and difficult to access via other technologies. In demonstration of this, we report a new high-quality, diploid aware de novo assembly of Craig Venter’s well-studied genome.


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