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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.

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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 one sequencing 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 typically requires high coverage for error correction, these benefits have in the past been lost for low-abundance species. The introduction of the Sequel II System has enabled a new, higher throughput, assembly-optional data type that addresses these challenges: HiFi reads. HiFi reads combine QV20 accuracy with long read lengths, eliminating the need for assembly for most metagenome applications, including gene discovery and metabolic pathway reconstruction. In fact, the read lengths and accuracy of HiFi data match or outperform the quality metrics of most metagenome assemblies, enabling cost-effective recovery of intact genes and operons while omitting the resource intensive and data-inefficient assembly step. Here we present the application of HiFi sequencing to both mock and human fecal samples using full-length 16S and shotgun methods. This proof-of-concept work demonstrates the unique strengths of the HiFi method. First, the high correspondence between the expected community composition,16S and shotgun profiling data reflects low context bias. In addition, every HiFi read yields ~5-8 predicted genes, without assembly, using standard tools. If assembly is desired, excellent results can be achieved with Canu and contig binning tools. In summary, HiFi sequencing is a new, cost-effective option for high-resolution functional profiling of metagenomes which complements existing short read workflows.

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June 1, 2021  |  

Copy-number variant detection with PacBio long reads

Long-read sequencing of diverse humans has revealed more than 20,000 insertion, deletion, and inversion structural variants spanning more than 12 Mb in a healthy human genome. Most of these variants are too large to detect with short reads and too small for array comparative genome hybridization (aCGH). While the standard approaches to calling structural variants with long reads thrive in the 50 bp to 10 kb size range, they tend to miss exactly the large (>50 kb) copy-number variants that are called more readily with aCGH. Standard algorithms rely on reference-based mapping of reads that fully span a variant or on de novo assembly; and copy-number variants are often too large to be spanned by a single read and frequently involve segmentally duplicated sequence that is not yet included in most de novo assemblies. To comprehensively detect large variants in human genomes, we extended pbsv – a structural variant caller for long reads – to call copy-number variants (CNVs) from read-clipping and read-depth signatures. In human germline benchmark samples, we detect more than 300 CNVs spanning around 10 Mb, and we call hundreds of additional events in re-arranged cancer samples. Together with insertion, deletion, inversion, duplication, and translocation calling from spanning reads, this allows pbsv to comprehensively detect large variants from a single data type.

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June 1, 2021  |  

Low-input single molecule HiFi sequencing for metagenomic samples

HiFi sequencing on the PacBio Sequel II System enables complete microbial community profiling of complex metagenomic samples using whole genome shotgun sequences. With HiFi sequencing, highly accurate long reads overcome the challenges posed by the presence of intergenic and extragenic repeat elements in microbial genomes, thus greatly improving phylogenetic profiling and sequence assembly. Recent improvements in library construction protocols enable HiFi sequencing starting from as low as 5 ng of input DNA. Here, we demonstrate comparative analyses of a control sample of known composition and a human fecal sample from varying amounts of input genomic DNA (1 ug, 200 ng, 5 ng), and present the corresponding library preparation workflows for standard, low input, and Ultra-Low methods. We demonstrate that the metagenome assembly, taxonomic assignment, and gene finding analyses are comparable across all methods for both samples, providing access to HiFi sequencing even for DNA-limited sample types.

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June 1, 2021  |  

Comprehensive variant detection in a human genome with highly accurate long reads

Introduction: Long-read sequencing has been applied successfully to assemble genomes and detect structural variants. However, due to high raw-read error rates (10-15%), it has remained difficult to call small variants from long reads. Recent improvements in library preparation and sequencing chemistry have increased length, accuracy, and throughput of PacBio circular consensus sequencing (CCS) reads, resulting in 15-20kb reads with average read quality above 99%. Materials and Methods: We sequenced a library from human reference sample HG002 to 18-fold coverage on the PacBio Sequel II with two SMRT Cells 8M. The CCS algorithm was used to generate highly accurate (average 99.9%) 12.9kb reads, which were mapped to the hg19 reference with pbmm2. We detected small variants using Google DeepVariant with a model trained for CCS and phased the variants using WhatsHap. Structural variants were detected with pbsv. Variant calls were evaluated against Genome in a Bottle (GIAB) benchmarks. Results: With these reads, DeepVariant achieves SNP and Indel F1 scores of 99.70% and 96.59% against the GIAB truth set, and pbsv achieves 97.72% recall on structural variants longer than 50bp. Using WhatsHap, small variants were phased into haplotype blocks with 145kb N50. The improved mappability of long reads allows us to align to and detect variants in medically relevant genes such as CYP2D6 and PMS2 that have proven “difficult-to-map” with short reads. Conclusions: These highly accurate long reads combine the mappability and ability to detect structural variants of long reads with the accuracy and ability to detect small variants of short reads.

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June 1, 2021  |  

A workflow for the comprehensive detection and prioritization of variants in human genomes with PacBio HiFi reads

PacBio HiFi reads (minimum 99% accuracy, 15-25 kb read length) have emerged as a powerful data type for comprehensive variant detection in human genomes. The HiFi read length extends confident mapping and variant calling to repetitive regions of the genome that are not accessible with short reads. Read length also improves detection of structural variants (SVs), with recall exceeding that of short reads by over 30%. High read quality allows for accurate single nucleotide variant and small indel detection, with precision and recall matching that of short reads. While many tools have been developed to take advantage of these qualities of HiFi reads, there is no end-to-end workflow for the filtering and prioritization of variants uniquely detected with long reads for rare and undiagnosed disease research. We have developed a flexible, modular workflow and web portal for variant analysis from HiFi reads and applied it to a set of rare disease cases unsolved by short-read whole genome sequencing. We expect that broad application of long-read variant detection workflows will solve many more rare disease cases. We have made these tools available at https://github.com/williamrowell/pbRUGD-workflow, and we hope they serve a starting point for developing a robust analysis framework for long read variant detection for rare diseases.

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June 1, 2021  |  

Comprehensive variant detection in a human genome with highly accurate long reads

Introduction: Long-read sequencing has revealed more than 20,000 structural variants spanning over 12 Mb in a healthy human genome. Short-read sequencing fails to detect most structural variants but has remained the more effective approach for small variants, due to 10-15% error rates in long reads, and copy-number variants (CNVs), due to lack of effective long-read variant callers. The development of PacBio highly accurate long reads (HiFi reads) with read lengths of 10-25 kb and quality >99% presents the opportunity to capture all classes of variation with one approach.Methods: We sequence the Genome in a Bottle benchmark sample HG002 and an individual with a presumed Mendelian disease with HiFi reads. We call SNVs and indels with DeepVariant and extend the structural variant caller pbsv to call CNVs using read depth and clipping signatures. Results: For 18-fold coverage with 13 kb HiFi reads, variant calling in HG002 achieves an F1 score of 99.7% for SNVs, 96.6% for indels, and 96.4% for structural variants. Additionally, we detect more than 300 CNVs spanning around 10 Mb. For the Mendelian disease case, HiFi reads reveal thousands of variants that were overlooked by short-read sequencing, including a candidate causative structural variant. Conclusions: These results illustrate the ability of HiFi reads to comprehensively detect variants, including those associated with human disease.

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June 1, 2021  |  

Amplification-free targeted enrichment powered by CRISPR-Cas9 and long-read Single Molecule Real-Time (SMRT) Sequencing can efficiently and accurately sequence challenging repeat expansion disorders

Genomic regions with extreme base composition bias and repetitive sequences have long proven challenging for targeted enrichment methods, as they rely upon some form of amplification. Similarly, most DNA sequencing technologies struggle to faithfully sequence regions of low complexity. This has been especially trying for repeat expansion disorders such as Fragile-X disease, Huntington disease and various Ataxias, where the repetitive elements range from several hundreds of bases to tens of kilobases. We have developed a robust, amplification-free targeted enrichment technique, called No-Amp Targeted Sequencing, that employs the CRISPR-Cas9 system. In conjunction with SMRT Sequencing, which delivers long reads spanning the entire repeat expansion, high consensus accuracy, and uniform coverage, these previously inaccessible regions are now accessible. This method is completely amplification-free, therefore removing any PCR errors and biases from the experiment. Furthermore, this technique also preserves native DNA molecules, allowing for direct detection and characterization of epigenetic signatures. The No-Amp method is a two-day protocol that is compatible with multiplexing of multiple targets and multiple samples in a single reaction, using as little as 1 µg of genomic DNA input per sample. We have successfully targeted a number of repeat expansion disorder loci including HTT, FMR1, C9orf7,2 as well as built an Ataxia panel which consists of 15 different disease-causing repeat expansion regions. Using the No-Amp method we have isolated hundreds of individual on-target molecules, allowing for reliable repeat size estimation, mosaicism detection and identification of interruption sequences with alleles as long as >2700 repeat unites ( >13 kb). In addition to multiplexing several targets, we have also multiplexed at least 20 samples in one experiment making the No-Amp Targeted Sequencing method a cost-effective option. Combining the CRISPR-Cas9 enrichment method with Single Molecule, Real-Time Sequencing provided us with base-level resolution of previously inaccessible regions of the genome, like disease-causing repeat expansions. No-Amp Targeted Sequencing captures, in one experiment, many aspects of repeat expansion disorders which are important for better understanding the underlying disease mechanisms.

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