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Tuesday, 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…

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Tuesday, June 1, 2021

A low DNA input protocol for high-quality PacBio de novo genome assemblies from single invertebrate individuals

A high-quality reference genome is an essential tool for studies of plant and animal genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. PacBio is the core technology for many large genome initiatives, however, relatively high DNA input requirements (5 µg for standard library protocol) have placed PacBio out of reach for many projects on small, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles…

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Tuesday, 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…

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Tuesday, June 1, 2021

A high-quality de novo genome assembly from a single mosquito using PacBio sequencing

A high-quality reference genome is an essential tool for studies of plant and animal genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. While PacBio is the core technology for many large genome initiatives, relatively high DNA input requirements (3 µg for standard library protocol) have placed PacBio out of reach for many projects on small, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles…

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Tuesday, June 1, 2021

A low DNA input protocol for high-quality PacBio de novo genome assemblies

A high-quality reference genome is an essential tool for studying the genetics of traits and disease, organismal, comparative and conservation biology, and population genomics. PacBio Single Molecule, Real-Time (SMRT) Sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives. However, relatively high DNA input requirements (3 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that may have lower DNA content…

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Tuesday, 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…

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Friday, February 5, 2021

Video: Overview of SMRT technology

PacBio’s SMRT technology harnesses the natural process of DNA replication, which is a highly efficient and accurate process. Our SMRT technology enables the observation of DNA synthesis as it occurs in real time.

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Friday, February 5, 2021

i5K Webinar: High-quality de novo insect genome assemblies using PacBio sequencing

PacBio Sequencing is characterized by very long sequence reads (averaging > 10,000 bases), lack of GC-bias, and high consensus accuracy. These features have allowed the method to provide a new gold standard in de novo genome assemblies, producing highly contiguous (contig N50 > 1 Mb) and accurate (> QV 50) genome assemblies. We will briefly describe the technology and then highlight the full workflow, from sample preparation through sequencing to data analysis, on examples of insect genome assemblies, and illustrate the difference these high-quality genomes represent with regard to biological insights, compared to fragmented draft assemblies generated by short-read sequencing.

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Friday, February 5, 2021

Webinar: SMRT Sequencing applications in plant and animal sciences: an overview

In this webinar, Emily Hatas of PacBio shares information about the applications and benefits of SMRT Sequencing in plant and animal biology, agriculture, and industrial research fields. This session contains an overview of several applications: whole-genome sequencing for de novo assembly; transcript isoform sequencing (Iso-Seq) method for genome annotation; targeted sequencing solutions; and metagenomics and microbial interactions. High-level workflows and best practices are discussed for key applications.

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Friday, February 5, 2021

Webinar: Understanding, curating, and analyzing your diploid genome assembly

The goal of this session is to help users complete their PacBio genome assembly and generate the best resource for their research. Kingan begins with a brief review of the diploid assembly process used by FALCON and FALCON-Unzip, highlighting the enhanced phasing of the Unzip module, and concluding with recommendations for genome polishing. Next, she explores how heterozygosity can influence the assembly process and how read coverage depth along the assembly can reveal important characteristics of assembly structure. Kingan then recommends approaches, including specific tools, that can be used to quality filter and curate the assembly, including annotation-, coverage-, and…

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Friday, February 5, 2021

Educational Video: Step 1 – from sample to sequencing data for de novo assembly

This video provides an overview of the techniques and steps of preparing samples, DNA, and libraries for PacBio Single Molecule, Real-Time (SMRT) Sequencing to be used in de novo assembly projects. In this video, a PacBio scientist covers how to assess DNA quantity and purity, size-selection of DNA libraries, and provides and introduction to SMRT Sequencing, including the benefits of long-reads when generating high-quality genome assemblies.

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Friday, February 5, 2021

Educational Video: Step 2 – de novo assembly of a genome

This video provides an overview of the techniques and steps of generating a de novo genome assembly with long-read sequencing data generated using PacBio Single Molecule, Real-Time (SMRT) Sequencing. In this video, a PacBio scientist covers the benefits of long reads when generating high-quality genome assemblies, the latest tools for creating assemblies, including HGAP, FALCON and FALCON-Unzip, how to polish and assess the quality of a genome assembly, and how to submit an assembly to NCBI.

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Friday, February 5, 2021

Webinar: A sketch of assembly recipes for PacBio data

This webinar, presented by Nisha Pillai, provides an overview of bioinformatics approaches for PacBio Single Molecule, Real-Time (SMRT) Sequencing data and discusses the whole genome sequencing application including: assembly workflow designs, an overview analysis tools for de novo assembly of SMRT Sequencing data (HGAP4, FALCON & FALCON-Unzip), and finally best practices and case studies.

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Friday, February 5, 2021

Webinar: Assembling high-quality human reference genomes for global populations

This webinar highlights global initiatives currently underway to use Single Molecule, Real-Time (SMRT) Sequencing to de novo assemble genomes of individuals representing multiple ethnic populations, thereby extending the diversity of available human reference genomes. In their presentations, Tina Graves-Lindsay from Washington University and Adam Ameur from Uppsala University spoke about diploid assemblies, discovering novel sequence and improving diversity of the current human reference genome. Finally, Paul Peluso of PacBio presented data from the recent effort to sequence a Puerto Rican genome and shared a SMRT Sequencing technology roadmap showing the next several upgrades for the Sequel System.

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