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Asset Tag: HiFi

June 1, 2021  |  

The value of long read amplicon sequencing for clinical applications

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. Homologous regions make NGS and MLPA results very difficult to interpret.

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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  |  

TLA & long-read sequencing: Efficient targeted sequencing and phasing of the CFTR gene

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 CFTR modulators. The Targeted Locus Amplification (TLA) technology from Cergentis can be used to selectively amplify, sequence and phase the entire CFTR gene. With PacBio long-read SMRT Sequencing, TLA amplicons are sequenced intact and long-range phasing information of all fragments in entire amplicons is retrieved. Experimental Design and Methods: The TLA process produces amplicons consisting of 5-10 proximity ligated DNA fragments. TLA was performed on cell line and genomic DNA from Coriell GM12878, which has few heterozygous SNVs in CFTR, and the IB3 cell line, with known haplotypes but heterozygous for the delta508 mutation. All sample types were prepared with high and low density TLA primer sets, targeting coverage of >100 kb of the CFTR gene. Conclusion: We have demonstrated the power and utility of TLA with long-read SMRT Sequencing as a valuable research tool in sequencing and phasing across very long regions of the human genome. This process can be done in an efficient manner, multiplexing multiple genes and samples per SMRT Cell in a process amenable to high-throughput sequencing.

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June 1, 2021  |  

Full-Length RNA-seq of Alzheimer brain on the PacBio Sequel II 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.

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June 1, 2021  |  

Comprehensive structural and copy-number variant detection with long reads

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 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 and short reads. 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.

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June 1, 2021  |  

Detection and phasing of small variants in Genome in a Bottle samples with highly accurate long reads

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 Sequel II System with six SMRT Cells 8M each. The CCS algorithm was used to generate highly accurate (average 99.8%) reads of mean length 10-11 kb, which were then mapped to the hs37d5 reference with pbmm2. We detected small variants using Google DeepVariant and compared these variant calls to GIAB benchmarks. Small variants were then phased with WhatsHap. Results: With these long, highly accurate CCS reads, DeepVariant achieves high SNP and Indel accuracy against the GIAB benchmark truth set for all three reference samples. Using WhatsHap, small variants were phased into haplotype blocks with N50 from 82 to 146 kb. The improved mappability of long reads allows detection of variants in many medically relevant genes such as CYP2D6and PMS2that have proven ‘difficult-to-map’ with short reads. We show that small variant precision and recall remain high down to 15-fold coverage. Conclusions: These highly accurate long reads combine the mappability of noisy long reads with the accuracy and small variant detection utility of short reads, which will allow the detection and phasing of variants in regions that have proven recalcitrant to short read sequencing and variant detection.

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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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June 1, 2021  |  

A complete solution for high-quality genome annotation using the PacBio Iso-Seq method

The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more transcripts. Most importantly, the method is supported by a maturing suite of official and community-developed tools. The SMRT Link Iso-Seq application outputs high-quality (>99% accurate), full-length transcript sequences that can optionally be mapped to a reference genome for a single SMRT Cell worth of data in 6-9 hours. For example, the SQANTI2 tool classifies Iso-Seq transcripts against a reference annotation, filters potential library artifacts, and processes information from both long read-only and short read-based quantification. IsoPhase is a tool for identifying allele-specific isoform expression. Cogent has been used to process Iso-Seq transcripts in a genome-independent manner to assess genome assemblies. Finally, IsoAnnot is an up-and-coming tool for identifying differential isoform expression across different samples. We describe how these tools complement each other and provide guidelines to make the best use out of Iso-Seq data for understanding transcriptomes.

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