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Friday, February 26, 2021

Best practices for diploid assembly of complex genomes using PacBio: A case study of Cascade Hops

A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over…

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Friday, February 26, 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…

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Friday, February 26, 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,…

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Friday, February 26, 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…

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Friday, February 26, 2021

Structural variant detection with long read sequencing reveals driver and passenger mutations in a melanoma cell line

Past large scale cancer genome sequencing efforts, including The Cancer Genome Atlas and the International Cancer Genome Consortium, have utilized short-read sequencing, which is well-suited for detecting single nucleotide variants (SNVs) but far less reliable for detecting variants larger than 20 base pairs, including insertions, deletions, duplications, inversions and translocations. Recent same-sample comparisons of short- and long-read human reference genome data have revealed that short-read resequencing typically uncovers only ~4,000 structural variants (SVs, =50 bp) per genome and is biased towards deletions, whereas sequencing with PacBio long-reads consistently finds ~20,000 SVs, evenly balanced between insertions and deletions. This discovery has…

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Friday, February 26, 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…

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Friday, February 26, 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…

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Friday, February 26, 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…

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Friday, February 26, 2021

Copy-number variant detection with PacBio long reads

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 healthy 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. Standard algorithms rely on reference-based mapping of reads that fully span a variant or…

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Friday, February 26, 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…

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Friday, February 26, 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%)…

Read More »

Friday, February 26, 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…

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Saturday, February 20, 2021

Application Brief: Variant detection using whole genome sequencing with HiFi reads – Best Practices

With highly accurate long reads (HiFi reads) from the Sequel II System, powered by Single Molecule, Real-Time (SMRT) Sequencing technology, you can comprehensively detect variants in a human genome. HiFi reads provide high precision and recall for single nucleotide variants (SNVs), indels, structural variants (SVs), and copy number variants (CNVs), including in difficult-to-map repetitive regions.

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