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June 1, 2021

Joint calling and PacBio SMRT Sequencing for indel and structural variant detection in populations

Fast and effective variant calling algorithms have been crucial to the successful application of DNA sequencing in human genetics. In particular, joint calling – in which reads from multiple individuals are pooled to increase power for shared variants – is an important tool for population surveys of variation. Joint calling was applied by the 1000 Genomes Project to identify variants across many individuals each sequenced to low coverage (about 5-fold). This approach successfully found common small variants, but broadly missed structural variants and large indels for which short-read sequencing has limited sensitivity. To support use of large variants in rare disease and common trait association studies, it is necessary to perform population-scale surveys with a technology effective at detecting indels and structural variants, such as PacBio SMRT Sequencing. For these studies, it is important to have a joint calling workflow that works with PacBio reads. We have developed pbsv, an indel and structural variant caller for PacBio reads, that provides a two-step joint calling workflow similar to that used to build the ExAC database. The first stage, discovery, is performed separately for each sample and consolidates whole genome alignments into a sparse representation of potentially variant loci. The second stage, calling, is performed on all samples together and considers only the signatures identified in the discovery stage. We applied the pbsv joint calling workflow to PacBio reads from twenty human genomes, with coverage ranging from 5-fold to 80-fold per sample for a total of 460-fold. The analysis required only 102 CPU hours, and identified over 800,000 indels and structural variants, including hundreds of inversions and translocations, many times more than discovered with short-read sequencing. The workflow is scalable to thousands of samples. The ongoing application of this workflow to thousands of samples will provide insight into the evolution and functional importance of large variants in human evolution and disease.

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June 1, 2021

A simple segue from Sanger to high-throughput SMRT Sequencing with a M13 barcoding system

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 generates read lengths shorter than the region of interest, which limits its ability to accurately phase allelic variants. High-throughput SMRT Sequencing overcomes the challenges of both the first- and second-generation sequencing methods. PacBio’s long read capability allows sequencing of full-length amplicons

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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

Improving the reference with a diversity panel of sequence-resolved structural variation

Although the accuracy of the human reference genome is critical for basic and clinical research, structural variants (SVs) have been difficult to assess because data capable of resolving them have been limited. To address potential bias, we sequenced a diversity panel of nine human genomes to high depth using long-read, single-molecule, real-time sequencing data. Systematically identifying and merging SVs =50 bp in length for these nine and one public genome yielded 83,909 sequence-resolved insertions, deletions, and inversions. Among these, 2,839 (2.0 Mbp) are shared among all discovery genomes with an additional 13,349 (6.9 Mbp) present in the majority of humans, indicating minor alleles or errors in the reference, which is partially explained by an enrichment for GC-content and repetitive DNA. Genotyping 83% of these in 290 additional genomes confirms that at least one allele of the most common SVs in unique euchromatin are now sequence-resolved. We observe a 9-fold increase within 5 Mbp of chromosome telomeric ends and correlation with de novo single-nucleotide variant mutations showing that such variation is nonrandomly distributed defining potential hotspots of mutation. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. To illustrate the utility of sequence-resolved SVs in resequencing experiments, we mapped 30 diverse high-coverage Illumina-sequenced samples to GRCh38 with and without contigs containing SV insertions as alternate sequences, and we found these additional sequences recover 6.4% of unmapped reads. For reads mapped within the SV insertion, 25.7% have a better mapping quality, and 18.7% improved by 1,000-fold or more. We reveal 72,964 occurrences of 15,814 unique variants that were not discoverable with the reference sequence alone, and we note that 7% of the insertions contain an SV in at least one sample indicating that there are additional alleles in the population that remain to be discovered. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity. We present a summary of our findings and discuss ideas for revealing variation that was once difficult to ascertain.

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June 1, 2021

Haplotyping using full-length transcript sequencing reveals allele-specific expression

An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with IsoPhase against matching short read data and identified cases of allele-specific, gene-level and isoform-level expression.

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June 1, 2021

Comprehensive variant detection in a human genome with PacBio high-fidelity reads

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, and Circular Consensus Sequence (CCS) reads, generated by calculating the consensus of many sequencing passes over a single shorter template (500 bp to 20 kbp). The long-range information in continuous reads is useful for genome assembly and structural variant detection. The higher base accuracy of CCS effectively detects and phases short variants in single molecules. Recent improvements in library preparation protocols and sequencing chemistry have increased the length, accuracy, and throughput of CCS reads. For the human sample HG002, we collected 28-fold coverage 15 kbp high-fidelity CCS reads with an average read quality above Q20 (99% accuracy). The length and accuracy of these reads allow us to detect SNVs, indels, and structural variants not only in the Genome in a Bottle (GIAB) high confidence regions, but also in segmental duplications, HLA loci, and clinically relevant “difficult-to-map” genes. As with continuous long reads, we call structural variants at 90.0% recall compared to the GIAB structural variant benchmark “truth” set, with the added advantages of base pair resolution for variant calls and improved recall at compound heterozygous loci. With minimap2 alignments, GATK4 HaplotypeCaller variant calls, and simple variant filtration, we have achieved a SNP F-Score of 99.51% and an INDEL F-Score of 80.10% against the GIAB short variant benchmark “truth” set, in addition to calling variants outside of the high confidence region established by GIAB using previous technologies. With the long-range information available in 15 kbp reads, we applied the read-backed phasing tool WhatsHap to generate phase blocks with a mean length of 65 kbp across the entire genome. Using an alignment-based approach, we typed all major MHC class I and class II genes to at least 3-field precision. This new data type has the potential to expand the GIAB high confidence regions and “truth” benchmark sets to many previously difficult-to-map genes and allow a single sequencing protocol to address both short variants and large structural variants.

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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

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 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 99.8%) 11.4kb 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 99.82% and 96.70% against the GIAB truth set, and pbsv achieves 95.94% recall on structural variants longer than 50bp. Using WhatsHap, small variants were phased into haplotype blocks with 105kb 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

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

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

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

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

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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