High-throughput NGS methods are increasingly utilized in the clinical genomics market. However, short-read sequencing data continues to remain challenged by mapping inaccuracies in low complexity regions or regions of high homology and may not provide adequate coverage within GC-rich regions of the genome. Thus, the use of Sanger sequencing remains popular in many clinical sequencing labs as the gold standard approach for orthogonal validation of variants and to interrogate missed regions poorly covered by second-generation sequencing. The use of Sanger sequencing can be less than ideal, as it can be costly for high volume assays and projects. Additionally, Sanger sequencing…
Human genomic variations range in size from single nucleotide substitutions to large chromosomal rearrangements. Sequencing technologies tend to be optimized for detecting particular variant types and sizes. Short reads excel at detecting SNVs and small indels, while long or linked reads are typically used to detect larger structural variants or phase distant loci. Long reads are more easily mapped to repetitive regions, but tend to have lower per-base accuracy, making it difficult to call short variants. The PacBio Sequel System produces two main data types: long continuous reads (up to 100 kbp), generated by single passes over a long template,…
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…
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 10-20kb reads with average read quality above 99%. Materials and Methods: We sequenced a 12kb library from human reference sample HG002 to 18-fold coverage on the PacBio Sequel II System with three SMRT Cells 8M. The CCS algorithm was used to generate highly-accurate (average…
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,…
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…
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 especially been true for repeat expansion disorders such as Fragile X syndrome, Huntington’s 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 Single Molecule, Real-Time (SMRT) Sequencing, which delivers…
Background: The sequencing and haplotype phasing of entire gene sequences improves the understanding of the genetic basis of disease and drug response. One example is cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have revolutionized CF treatment, but only in a minority of CF subjects. Observed heterogeneity in CFTR modulator efficacy is related to the range of CFTR mutations; revertant mutations can modify the response to CFTR modulators, and other intronic variations in the ~200 kb CFTR gene have been linked to disease severity. Heterogeneity in the CFTR gene may also be linked to differential responses to…
NGS is commonly used for amplicon sequencing in clinical applications to study genetic disorders and detect disease-causing mutations. This approach can be plagued by limited ability to phase sequence variants and makes interpretation of sequence data difficult when pseudogenes are present. Long-read highly accurate amplicon sequencing can provide very accurate, efficient, high throughput (through multiplexing) sequences from single molecules, with read lengths largely limited by PCR. Data is easy to interpret; phased variants and breakpoints are present within high fidelity individual reads. Here we show SMRT Sequencing of the PMS2 and OPN1 (MW and LW) genes using the Sequel System.…
The PacBio Iso-Seq method produces high-quality, full-length transcripts and can characterize a whole transcriptome with a single SMRT Cell 8M. We sequenced an Alzheimer whole brain sample on a single SMRT Cell 8M on the Sequel II System. Using the Iso-Seq bioinformatics pipeline followed by SQANTI2 analysis, we detected 162,290 transcripts for 17,670 genes up to 14 kb in length. More than 60% of the transcripts are novel isoforms, the vast majority of which have supporting cage peak data and polyadenylation signals, demonstrating the utility of long-read sequencing for human disease research.
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…
Introduction: Long-read PacBio SMRT Sequencing has been applied successfully to assemble genomes and detect structural variants. However, due to high raw read error rates of 10-15%, it has remained difficult to call small variants from long reads. Recent improvements in library preparation, sequencing chemistry, and instrument yield have increased length, accuracy, and throughput of PacBio Circular Consensus (CCS) reads, resulting in 10-20 kb “HiFi” reads with mean read quality above 99%. Materials and Methods: We sequenced 11 kb size-selected libraries from the Genome in a Bottle (GIAB) human reference samples HG001, HG002, and HG005 to approximately 30-fold coverage on the…
To comprehensively detect large variants in human genomes, we have 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. 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 typical human genome. Most of these variants are too large to detect with short reads and too small for array…
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,…
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…