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Asset Tag: long-read sequencing

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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May 17, 2021

Genomic Answers for Kids

Short-read genome-wide sequencing for molecular diagnosis has revolutionized pediatric rare disease care in the past decade. However, most families remain without specific knowledge of the cause of their child’s illness….

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