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June 1, 2021  |  

Allelic specificity of immunoglobulin heavy chain (IGH@) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).

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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.

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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.

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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.

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June 1, 2021  |  

A low DNA input protocol for high-quality PacBio de novo genome assemblies from single invertebrate individuals

A high-quality reference genome is an essential tool for studies of plant and animal 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. PacBio is the core technology for many large genome initiatives, however, relatively high DNA input requirements (5 µg for standard library protocol) have placed PacBio out of reach for many projects on small, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles coluzzii mosquito and the Schistosoma mansoni parasitic flatworm. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 50-100 ng of starting genomic DNA. The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating a range of 21-32 Gb of sequence per SMRT Cell with 20 hour movies, and followed by diploid de novo genome assembly with FALCON-Unzip. The resulting assemblies had high contiguity (contig N50s over 3 Mb for both species) and completeness (as determined by conserved BUSCO gene analysis). We were also able to resolve maternal and paternal haplotypes for 1/3 of the genome in both cases. 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. This new low-input approach puts PacBio-based assemblies in reach for small, highly heterozygous organisms that comprise much of the diversity of life. The method presented here can be applied to samples with starting DNA amounts around 100 ng per 250 Mb – 1 Gb genome size.

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June 1, 2021  |  

Single molecule high-fidelity (HiFi) Sequencing with >10 kb libraries

Recent improvements in sequencing chemistry and instrument performance combine to create a new PacBio data type, Single Molecule High-Fidelity reads (HiFi reads). Increased read length and improvement in library construction enables average read lengths of 10-20 kb with average sequence identity greater than 99% from raw single molecule reads. The resulting reads have the accuracy comparable to short read NGS but with 50-100 times longer read length. Here we benchmark the performance of this data type by sequencing and genotyping the Genome in a Bottle (GIAB) HG0002 human reference sample from the National Institute of Standards and Technology (NIST). We further demonstrate the general utility of HiFi reads by analyzing multiple clones of Cabernet Sauvignon. Three different clones were sequenced and de novo assembled with the CANU assembly algorithm, generating draft assemblies of very high contiguity equal to or better than earlier assembly efforts using PacBio long reads. Using the Cabernet Sauvignon Clone 8 assembly as a reference, we mapped the HiFi reads generated from Clone 6 and Clone 47 to identify single nucleotide polymorphisms (SNPs) and structural variants (SVs) that are specific to each of the three samples.

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June 1, 2021  |  

A high-quality de novo genome assembly from a single mosquito using PacBio sequencing

A high-quality reference genome is an essential tool for studies of plant and animal 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. While PacBio is the core technology for many large genome initiatives, relatively high DNA input requirements (3 µg for standard library protocol) have placed PacBio out of reach for many projects on small, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles coluzzii mosquito and the Schistosoma mansoni parasitic flatworm. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 150 ng of starting genomic DNA. The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating a range of 21-32 Gb of sequence per SMRT Cell with 20-hour movies (10-12 Gb for 10-hour movies), and followed by diploid de novo genome assembly with FALCON-Unzip. The resulting assemblies had high contiguity (contig N50s over 3 Mb for both species) and completeness (as determined by conserved BUSCO gene analysis). We were also able to resolve maternal and paternal haplotypes for 1/3 of the genome in both cases. 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. This new low-input approach puts PacBio-based assemblies in reach for small, highly heterozygous organisms that comprise much of the diversity of life. The method presented here can be applied to samples with starting DNA amounts around 150 ng per 250 Mb – 600 Mb genome size.

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June 1, 2021  |  

Streamlines SMRTbell library generation using addition-only, single tube strategy for all library types reduces time to results

We have streamlined the SMRTbell library generation protocols with improved workflows to deliver seamless end-to-end solutions from sample to analysis. A key improvement is the development of a single-tube reaction strategy that shortened hands-on time needed to generate each SMRTbell library, reduced time-consuming AM Pure purification steps, and minimized sample-handling induced gDNA damage to improve the integrity of long-insert SMRTbell templates for sequencing. The improved protocols support all large-insert genomic libraries, multiplexed microbial genomes, and amplicon sequencing. These advances enable completion of library preparation in less than a day (approximately 4 hours) and opens opportunities for automated library preparation for large-scale projects. Here we share data summarizing performance of the new SMRTbell Express Template Kit 2.0 representing our solutions for 10 kb and >50 kb large-insert genomic libraries, complete microbial genome assemblies, and high-throughput amplicon sequencing. The improved throughput of the Sequel System with read lengths up to 30 kb and high consensus accuracy (> 99.999% accuracy) makes sequencing with high-quality results increasingly assessible to the community.

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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.

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June 1, 2021  |  

Comparison of sequencing approaches applied to complex soil metagenomes to resolve proteins of interest

Background: Long-read sequencing presents several potential advantages for providing more complete gene profiling of metagenomic samples. Long reads can capture multiple genes in a single read, and longer reads typically result in assemblies with better contiguity, especially for higher abundance organisms. However, a major challenge with using long reads has been the higher cost per base, which may lead to insufficient coverage of low-abundance species. Additionally, lower single-pass accuracy can make gene discovery for low-abundance organisms difficult. Methods: To evaluate the pros and cons of long reads for metagenomics, we directly compared PacBio and Illumina sequencing on a soil-derived sample, which included spike-in controls of known concentrations of pure referenced samples. For PacBio sequencing, a 10 kb library was sequenced on the Sequel System with 3.0 chemistry. Highly accurate long reads (HiFi reads) with Q20 and higher were generated for downstream analyses using PacBio Circular Consensus Sequencing (CCS) mode. Results were assessed according to the following criteria: DNA extraction capacity, bioinformatics pipeline status, % of proteins with ambiguous AA’s, total unique error-free genes/$1000, total proteins observed in spike-ins/$1000, proteins of interest/$1000, median length of contigs with proteins, and assembly requirements. Results: Both methods had areas of superior performance. DNA extraction capacity was higher for Illumina, the bioinformatics pipeline is well-tested, and there was a lower proportion of proteins with ambiguous AA’s. On the other hand, with PacBio, twice as many unique error-free genes, twice as many total proteins from spike-ins, and ~6 times more proteins of interest were found per $1000 cost. PacBio data produced on average 5 times longer contigs capturing proteins of interest. Additionally, assembly was not required for gene or protein finding, as was the case with Illumina data. Conclusions: In this comparison of PacBio Sequel System with Illumina NextSeq on a complex microbiome, we conclude that the sequencing system of choice may vary, depending on the goals and resources for the project. PacBio sequencing requires a longer DNA extraction method, and the bioinformatics pipeline may require development. On the other hand, the Sequel System generates hundreds of thousands of long HiFi reads per SMRT Cell, producing more genes, more proteins, and longer contigs, thereby offering more information about the metagenomic samples for a lower cost.

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June 1, 2021  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with short-read technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly requires high coverage for error correction, the benefits of unbiased coverage have in the past been lost for low abundance species. SMRT Sequencing performance improvements and the introduction of the Sequel II System has enabled a new, high throughput data type uniquely suited to metagenome characterization: HiFi reads. HiFi reads combine high accuracy with read lengths up to 15 kb, eliminating the need for assembly for most microbiome applications, including functional profiling, gene discovery, and metabolic pathway reconstruction. Here we present the application of the HiFi data type to enable a new method of analyzing metagenomes that does not require assembly.

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June 1, 2021  |  

High-quality human genomes achieved through HiFi sequence data and FALCON-Unzip assembly

De novo assemblies of human genomes from accurate (85-90%), continuous long reads (CLR) now approach the human reference genome in contiguity, but the assembly base pair accuracy is typically below QV40 (99.99%), an order-of-magnitude lower than the standard for finished references. The base pair errors complicate downstream interpretation, particularly false positive indels that lead to false gene loss through frameshifts. PacBio HiFi sequence data, which are both long (>10 kb) and very accurate (>99.9%) at the individual sequence read level, enable a new paradigm in human genome assembly. Haploid human assemblies using HiFi data achieve similar contiguity to those using CLR data and are highly accurate at the base level1. Furthermore, HiFi assemblies resolve more high-identity sequences such as segmental duplications2. To enable HiFi assembly in diploid human samples, we have extended the FALCON-Unzip assembler to work directly with HiFi reads. Here we present phased human diploid genome assemblies from HiFi sequencing of HG002, HG005, and the Vertebrate Genome Project (VGP) mHomSap1 trio on the PacBio Sequel II System. The HiFi assemblies all exceed the VGP’s quality guidelines, approaching QV50 (99.999%) accuracy. For HG002, 60% of the genome was haplotype-resolved, with phase-block N50 of 143Kbp and phasing accuracy of 99.6%. The overall mean base accuracy of the assembly was QV49.7. In conclusion, HiFi data show great promise towards complete, contiguous, and accurate diploid human assemblies.

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June 1, 2021  |  

Every species can be a model: Reference-quality PacBio genomes from single insects

A high-quality reference genome is an essential resource for primary and applied research across the tree of life. Genome projects for small-bodied, non-model organisms such as insects face several unique challenges including limited DNA input quantities, high heterozygosity, and difficulty of culturing or inbreeding in the lab. Recent progress in PacBio library preparation protocols, sequencing throughput, and read accuracy address these challenges. We present several case studies including the Red Admiral (Vanessa atalanta), Monarch Butterfly (Danaus plexippus), and Anopheles malaria mosquitoes that highlight the benefits of sequencing single individuals for de novo genome assembly projects, and the ease at which these projects can be conducted by individual research labs. Sampled individuals may originate from lab colonies of interest to the research community or be sourced from the wild to better capture natural variation in a focal population. Where genomic DNA quantities are limited, the PacBio Low DNA Input Protocol requires ~100 ng of input DNA. Low DNA input samples with 500 Mb genome size or less can be multiplexed on a single SMRT Cell 8M on the Sequel II System. For samples with more abundant DNA quantity, size-selected libraries may be constructed to maximize sequencing yield. Both low DNA input and size-selected libraries can be used to generate HiFi reads, whose quality is Q20 or above (1% error or less) and lengths range from 10 – 25 kb. With HiFi reads, de novo assembly computation is greatly simplified relative to long read methods due to smaller sequence file sizes and more rapid analysis, resulting in highly accurate, contiguous, complete, and haplotype-resolved assemblies.

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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.

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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.

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