Menu

Asset Tag: de novo assembly

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.

Read more
June 1, 2021

De novo assembly and preliminary annotation of the Schizocardium californicum genome

Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.

Read more
June 1, 2021

Characterizing the pan-genome of maize with PacBio SMRT Sequencing

Maize is an amazingly diverse crop. A study in 20051 demonstrated that half of the genome sequence and one-third of the gene content between two inbred lines of maize were not shared. This diversity, which is more than two orders of magnitude larger than the diversity found between humans and chimpanzees, highlights the inability of a single reference genome to represent the full pan-genome of maize and all its variants. Here we present and review several efforts to characterize the complete diversity within maize using the highly accurate long reads of PacBio Single Molecule, Real-Time (SMRT) Sequencing. These methods provide a framework for a pan-genomic approach that can be applied to studies of a wide variety of important crop species.

Read more
June 1, 2021

Multiplexed complete microbial genomes on the Sequel System

Microbes play an important role in nearly every part of our world, as they affect human health, our environment, agriculture, and aid in waste management. Complete closed genome sequences, which have become the gold standard with PacBio long-read sequencing, can be key to understanding microbial functional characteristics. However, input requirements, consumables costs, and the labor required to prepare and sequence a microbial genome have in the past put PacBio sequencing out of reach for some larger projects. We have developed a multiplexed library prep approach that is simple, fast, and cost-effective, and can produce 4 to 16 closed bacterial genomes from one Sequel SMRT Cell. Additionally, we are introducing a streamlined analysis pipeline for processing multiplexed genome sequence data through de novo HGAP assembly, making the entire process easy for lab personnel to perform. Here we present the entire workflow from shearing through assembly, with times for each step. We show HGAP assembly results with single or very few contigs from bacteria from different size genomes, sequenced without or with size selection. These data illustrate the benefits and potential of the PacBio multiplexed library prep and the Sequel System for sequencing large numbers of microbial genomes.

Read more
June 1, 2021

High-quality de novo genome assembly and intra-individual mitochondrial instability in the critically endangered kakapo

The kakapo (Strigops habroptila) is a large, flightless parrot endemic to New Zealand. It is highly endangered with only ~150 individuals remaining, and intensive conservation efforts are underway to save this iconic species from extinction. These include genetic studies to understand critical genes relevant to fertility, adaptation and disease resistance, and genetic diversity across the remaining population for future breeding program decisions. To aid with these efforts, we have generated a high-quality de novo genome assembly using PacBio long-read sequencing. Using the new diploid-aware FALCON-Unzip assembler, the resulting genome of 1.06 Gb has a contig N50 of 5.6 Mb (largest contig 29.3 Mb), >350-times more contiguous compared to a recent short-read assembly of a closely related parrot (kea) species. We highlight the benefits of the higher contiguity and greater completeness of the kakapo genome assembly through examples of fully resolved genes important in wildlife conservation (contrasted with fragmented and incomplete gene resolution in short-read assemblies), in some cases even providing sequence for regions orthologous to gaps of missing sequence in the chicken reference genome. We also highlight the complete resolution of the kakapo mitochondrial genome, fully containing the mitochondrial control region which is missing from the previous dedicated kakapomitochondrial genome NCBI entry. For this region, we observed a marked heterogeneity in the number of tandem repeats in different mtDNAmolecules from a single bird tissue, highlighting the enhanced molecular resolution uniquely afforded by long-read, single-molecule PacBio sequencing.

Read more
June 1, 2021

Best practices for whole genome sequencing using the Sequel System

Plant and animal whole genome sequencing has proven to be challenging, particularly due to genome size, high density of repetitive elements and heterozygosity. The Sequel System delivers long reads, high consensus accuracy and uniform coverage, enabling more complete, accurate, and contiguous assemblies of these large complex genomes. The latest Sequel chemistry increases yield up to 8 Gb per SMRT Cell for long insert libraries >20 kb and up to 10 Gb per SMRT Cell for libraries >40 kb. In addition, the recently released SMRTbell Express Template Prep Kit reduces the time (~3 hours) and DNA input (~3 µg), making the workflow easy to use for multi- SMRT Cell projects. Here, we recommend the best practices for whole genome sequencing and de novo assembly of complex plant and animal genomes. Guidelines for constructing large-insert SMRTbell libraries (>30 kb) to generate optimal read lengths and yields using the latest Sequel chemistry are presented. We also describe ways to maximize library yield per preparation from as littles as 3 µg of sheared genomic DNA. The combination of these advances makes plant and animal whole genome sequencing a practical application of the Sequel System.

Read more
June 1, 2021

Single chromosomal genome assemblies on the Sequel System with Circulomics high molecular weight DNA extraction for microbes

Background: The Nanobind technology from Circulomics provides an elegant HMW DNA extraction solution for genome sequencing of Gram-positive and -negative microbes. Nanobind is a nanostructured magnetic disk that can be used for rapid extraction of high molecular weight (HMW) DNA from diverse sample types including cultured cells, blood, plant nuclei, and bacteria. Processing can be completed in <1 hour for most sample types and can be performed manually or automated with common instruments. Methods:We have validated several critical steps for generating high-quality microbial genome assemblies in a streamlined microbial multiplexing workflow. This new workflow enables high-volume, cost-effective sequencing of up to 16 microbes totaling 30 Mb in genome size on a single SMRT Cell 1M using a target shear size of 10 kb. We also evaluated this method on a pool of four “class 3” microbes that contain >7 kb repeats. Fragment size was increased to ~14 kb, with some fragments >30 kb. Results: Here we present a demonstration of these capabilities using isolates relevant to high-throughput sequencing applications, including common foodborne pathogens (Shigella, Listeria, Salmonella), and species often seen in hospital settings (Klebsiella, Staphylococcus). For nearly all microbes, including difficult-to-assemble class III microbes, we achieved complete de novo microbial assemblies of =5 chromosomal contigs with minimum quality scores of 40 (99.99% accuracy) using data from multiplexed SMRTbell libraries. Each library was sequenced on a single SMRT Cell 1M with the PacBio Sequel System and analyzed with streamlined SMRT Analysis assembly methods. Conclusions: We achieved high-quality, closed microbial genomes using a combination of Circulomics Nanobind extraction and PacBio SMRT Sequencing, along with a newly streamlined workflow that includes automated demultiplexing and push-button assembly.

Read more
June 1, 2021

FALCON-Phase integrates PacBio and HiC data for de novo assembly, scaffolding and phasing of a diploid Puerto Rican genome (HG00733)

Haplotype-resolved genomes are important for understanding how combinations of variants impact phenotypes. The study of disease, quantitative traits, forensics, and organ donor matching are aided by phased genomes. Phase is commonly resolved using familial data, population-based imputation, or by isolating and sequencing single haplotypes using fosmids, BACs, or haploid tissues. Because these methods can be prohibitively expensive, or samples may not be available, alternative approaches are required. de novo genome assembly with PacBio Single Molecule, Real-Time (SMRT) data produces highly contiguous, accurate assemblies. For non-inbred samples, including humans, the separate resolution of haplotypes results in higher base accuracy and more contiguous assembled sequences. Two primary methods exist for phased diploid genome assembly. The first, TrioCanu requires Illumina data from parents and PacBio data from the offspring. The long reads from the child are partitioned into maternal and paternal bins using parent-specific sequences; the separate PacBio read bins are then assembled, generating two fully phased genomes. An alternative approach (FALCON-Unzip) does not require parental information and separates PacBio reads, during genome assembly, using heterozygous SNPs. The length of haplotype phase blocks in FALCON-Unzip is limited by the magnitude and distribution of heterozygosity, the length of sequence reads, and read coverage. Because of this, FALCON-Unzip contigs typically contain haplotype-switch errors between phase blocks, resulting in primary contig of mixed parental origin. We developed FALCON-Phase, which integrates Hi-C data downstream of FALCON-Unzip to resolve phase switches along contigs. We applied the method to a human (Puerto Rican, HG00733) and non-human genome assemblies and evaluated accuracy using samples with trio data. In a cattle genome, we observe >96% accuracy in phasing when compared to TrioCanu assemblies as well as parental SNPs. For a high-quality PacBio assembly (>90-fold Sequel coverage) of a Puerto Rican individual we scaffolded the FALCON-Phase contigs, and re-phased the contigs creating a de novo scaffolded, phased diploid assembly with chromosome-scale contiguity.

Read more
June 1, 2021

A low DNA input protocol for high-quality PacBio de novo genome assemblies

A high-quality reference genome is an essential tool for studying the genetics of traits and disease, organismal, comparative and conservation biology, and population genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives. However, relatively high DNA input requirements (3 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that may have lower DNA content or on projects with limited input DNA for other reasons. Here we present a modified SMRTbell library construction protocol without DNA shearing or size selection that can be used to generate a SMRTbell library from just 150 ng of starting genomic DNA. Remarkably, the protocol enables high quality de novo assemblies from single invertebrate individuals and is applied to taxonomically diverse samples. By sequencing and assembling material from a single diploid individual, only two haplotypes are present, simplifying the assembly process compared to samples from multiple pooled individuals. The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating ~11 Gb of sequence per SMRT Cell with 10 hour movies, and followed by de novo genome assembly with FALCON. The resulting assemblies had high contiguity (contig N50s over 1 Mb) and completeness (as determined by conserved BUSCO gene analysis) when at least 30-fold unique molecular coverage is obtained. This new low-input approach now puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life. The method presented here is scalable and can be applied to samples with starting DNA amounts of 150 ng per 300 Mb genome size.

Read more
June 1, 2021

Beyond Contiguity: Evaluating the accuracy of de novo genome assemblies

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, but presents a challenge that any new benchmark must significantly exceed Q50 or the discordance will represent the error rate of the benchmark. To establish benchmarks for Oryza sativa and Drosophila melanogaster, we collected draft references, Illumina short reads, and PacBio HiFi reads. By species, the benchmark was defined as regions of normal coverage that are not within 5 bp of a small variant or 50 bp of a structural variant. For both species, the benchmark regions span around 60% of the genome and HiFi assemblies achieve Q50 accuracy, which is notably more accurate than assemblies with other technologies and meets typical standards for a finished, reference-grade assembly. Here we present a protocol to generate benchmarks for any sample that rival the GIAB benchmark in accuracy. These benchmarks allow the comparison and improvement of genome assemblies and highlight the superior accuracy of assemblies generated with PacBio HiFi reads.

Read more
June 1, 2021

A high-quality PacBio insect genome from 5 ng of input DNA

High-quality insect genomes are essential resources to understand insect biology and to combat them as disease vectors and agricultural pests. It is desirable to sequence a single individual for a reference genome to avoid complications from multiple alleles during de novo assembly. However, the small body size of many insects poses a challenge for the use of long-read sequencing technologies which often have high DNA-input requirements. The previously described PacBio Low DNA Input Protocol starts with ~100 ng of DNA and allows for high-quality assemblies of single mosquitoes among others and represents a significant step in reducing such requirements. Here, we describe a new library protocol with a further 20-fold reduction in the DNA input quantity. Starting with just 5 ng of high molecular weight DNA, we describe the successful sequencing and de novo genome assembly of a single male sandfly (Phlebotomus papatasi, the main vector of the Old World cutaneous leishmaniasis), using HiFi data generated on the PacBio Sequel II System and assembled with FALCON. The assembly shows a high degree of completeness (>97% of BUSCO genes are complete), contiguity (contig N50 of 1 Mb), and sequence accuracy (>98% of BUSCO genes without frameshift errors). This workflow has general utility for small-bodied insects and other plant and animal species for both focused research studies or in conjunction with large-scale genome projects.

Read more
June 1, 2021

New advances in SMRT Sequencing facilitate multiplexing for de novo and structural variant studies

The latest advancements in Sequel II SMRT Sequencing have increased average read lengths up to 50% compared to Sequel II chemistry 1.0 which allows multiplexing of 2-3 small organisms (<500 Mb) such as insects and worms for producing reference quality assemblies, calling structural variants for up to 2 samples with ~3 Gb genomes, analysis of 48 microbial genomes, and up to 8 communities for metagenomic profiling in a single SMRT Cell 8M. With the improved processivity of the new Sequel II sequencing polymerase, more SMRTbell molecules reach rolling circle mode resulting in longer overall read lengths, thus allowing efficient detection of barcodes (up to 80%) in the SMRTbell templates. Multiplexing of genomes larger than microbial organisms is now achievable. In collaboration with the Wellcome Sanger Institute, we have developed a workflow for multiplexing two individual Anopheles coluzzii using as low as 150 ng genomic DNA per individual. The resulting assemblies had high contiguity (contig N50s over 3 Mb) and completeness (>98% of conserved genes) for both individuals. For microbial multiplexing, we multiplexed 48 microbes with varying complexities and sizes ranging 1.6-8.0 Mb in single SMRT Cell 8M. Using a new end-to-end analysis (Microbial Assembly Analysis, SMRT Link 8.0), assemblies resulted in complete circularized genomes (>200-fold coverage) and efficient detection of >3-200 kb plasmids. Finally, the long read lengths (>90 kb) allows detection of barcodes in large insert SMRTbell templates (>15 kb) thus facilitating multiplex of two human samples in 1 SMRT Cell 8M for detecting SVs, Indels and CNVs. Here, we present results and describe workflows for multiplexing samples for specific applications for SMRT Sequencing.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.