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June 1, 2021  |  

Amplification-free targeted enrichment and SMRT Sequencing of repeat-expansion genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods are reliant upon some form of amplification. Amplification removes the epigenetic marks present in native DNA, and some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, a large number of genetic disorders are caused by expansions of repeat sequences. Furthermore, for some disorders, methylation status has been shown to be a key factor in the mechanism of disease.

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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  |  

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be economical for obtaining sequence information for defined regions of the genome. However, most target enrichment methods are reliant upon some form of amplification which can negatively impact downstream analysis. For example, amplification removes epigenetic marks present in native DNA, including nucleotide methylation, which are hypothesized to contribute to disease mechanisms in some disorders. In addition, some genomic regions known to be causative of many genetic disorders have extreme GC content and/or repetitive sequences that tend to be recalcitrant to faithful amplification. We have developed a novel, amplification-free enrichment technique that employs the CRISPR/Cas9 system to target individual genes. This method, in conjunction with the long reads, high consensus accuracy, and uniform coverage of SMRT Sequencing, allows accurate sequence analysis of complex genomic regions that cannot be investigated with other technologies. Using this strategy, we have successfully targeted a number of repeat expansion disorder loci (HTT, FMR1, ATXN10, C9orf72).With this data, we demonstrate the ability to isolate thousands of individual on-target molecules and, using the Sequel System, accurately sequence through long repeats regardless of the extreme GC-content. The method is compatible with multiplexing of multiple target loci and multiple samples in a single reaction. Furthermore, because there is no amplification step, this technique also preserves native DNA molecules for sequencing, allowing for the direct detection and characterization of epigenetic signatures. To this end, we demonstrate the detection of 5-mC in the CGG repeat of the FMR1 gene that is responsible for Fragile X syndrome.

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June 1, 2021  |  

No-amp targeted SMRT sequencing using a CRISPR-Cas9 enrichment method

Targeted sequencing of genomic DNA requires an enrichment method to generate detectable amounts of sequencing products. Genomic regions with extreme composition bias and repetitive sequences can pose a significant enrichment challenge. Many genetic diseases caused by repeat element expansions are representative of these challenging enrichment targets. PCR amplification, used either alone or in combination with a hybridization capture method, is a common approach for target enrichment. While PCR amplification can be used successfully with genomic regions of moderate to high complexity, it is the low-complexity regions and regions containing repetitive elements sometimes of indeterminate lengths due to repeat expansions that can lead to poor or failed PCR enrichment. We have developed an enrichment method for targeted SMRT Sequencing on the PacBio Sequel System using the CRISPR-Cas9 system that requires no PCR amplification. Briefly, a preformed SMRTbell library containing the target region of interest is cleaved with Cas9 through direct interaction with a sequence-specific guide RNA. After ligation with new poly(A) hairpin adapters, the asymmetric SMRTbell templates are enriched by magnetic bead separation. This method, paired with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows sequencing of genomic regions regardless of challenging sequence context that cannot be investigated with other technologies. The method is amenable to analyzing multiple samples and/or targets in a single reaction. In addition, this method also preserves epigenetic modifications allowing for the detection and characterization of DNA methylation which has been shown to be a key factor in the disease mechanism for some repeat expansion diseases. Here we present results of our latest No-Amp Targeted Sequencing procedure applied to the characterization of CAG triplet repeat expansions in the HTT gene responsible for Huntington’s Disease.

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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  |  

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  |  

Sequencing the previously unsequenceable using amplification-free targeted enrichment powered by CRISPR/Cas9

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 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, compatible with multiplexing of multiple targets and 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 (HTT, FMR1, ATXN10, C9orf72) with alleles as long as >2700 repeat unites (>13 kb). 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 – all aspects of repeat expansion disorders which are important for better understanding the underlying disease mechanisms.

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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  |  

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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