Learn how highly accurate long-read sequencing from the Sequel IIe Systems delivers data you can trust for advanced biological insights across a range of applications.
Scientists from WashU, Macrogen, and Mount Sinai are using long-read sequencing with single-molecule, next-generation genome mapping to create gold-quality de novo assemblies of human genomes. Unbiased de novo assembled genomes also highlight the substantial amount of structural variation unique to individuals and populations, which cannot be accessed by short-read technologies that use a reference-based re-sequencing approach.
After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38, along…
Completing a genome is an important goal of genome assembly. However, many assemblies, including reference assemblies, are unfinished and have a number of gaps. Long reads obtained from third-generation sequencing (TGS) platforms can help close these gaps and improve assembly contiguity. However, current gap-closure approaches using long reads require extensive runtime and high memory usage. Thus, a fast and memory-efficient approach using long reads is needed to obtain complete genomes.We developed LR_Gapcloser to rapidly and efficiently close the gaps in genome assembly. This tool utilizes long reads generated from TGS sequencing platforms. Tested on de novo assembled gaps, repeat-derived gaps,…
Contaminant sequences that appear in published genomes can cause numerous problems for downstream analyses, particularly for evolutionary studies and metagenomics projects. Our large-scale scan of complete and draft bacterial and archaeal genomes in the NCBI RefSeq database reveals that 2250 genomes are contaminated by human sequence. The contaminant sequences derive primarily from high-copy human repeat regions, which themselves are not adequately represented in the current human reference genome, GRCh38. The absence of the sequences from the human assembly offers a likely explanation for their presence in bacterial assemblies. In some cases, the contaminating contigs have been erroneously annotated as containing…
We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that…
Accurate genome assembly is hampered by repetitive regions. Although long single molecule sequencing reads are better able to resolve genomic repeats than short-read data, most long-read assembly algorithms do not provide the repeat characterization necessary for producing optimal assemblies. Here, we present Flye, a long-read assembly algorithm that generates arbitrary paths in an unknown repeat graph, called disjointigs, and constructs an accurate repeat graph from these error-riddled disjointigs. We benchmark Flye against five state-of-the-art assemblers and show that it generates better or comparable assemblies, while being an order of magnitude faster. Flye nearly doubled the contiguity of the human genome…
The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5?kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions 15?megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.
Rapid innovation in sequencing technologies and improvement in assembly algorithms have enabled the creation of highly contiguous mammalian genomes. Here we report a chromosome-level assembly of the water buffalo (Bubalus bubalis) genome using single-molecule sequencing and chromatin conformation capture data. PacBio Sequel reads, with a mean length of 11.5?kb, helped to resolve repetitive elements and generate sequence contiguity. All five B. bubalis sub-metacentric chromosomes were correctly scaffolded with centromeres spanned. Although the index animal was partly inbred, 58% of the genome was haplotype-phased by FALCON-Unzip. This new reference genome improves the contig N50 of the previous short-read based buffalo assembly…
Current genotyping approaches for single-nucleotide variations rely on short, accurate reads from second-generation sequencing devices. Presently, third-generation sequencing platforms are rapidly becoming more widespread, yet approaches for leveraging their long but error-prone reads for genotyping are lacking. Here, we introduce a novel statistical framework for the joint inference of haplotypes and genotypes from noisy long reads, which we term diplotyping. Our technique takes full advantage of linkage information provided by long reads. We validate hundreds of thousands of candidate variants that have not yet been included in the high-confidence reference set of the Genome-in-a-Bottle effort.
In recent genome analyses, population-specific reference panels have indicated important. However, reference panels based on short-read sequencing data do not sufficiently cover long insertions. Therefore, the nature of long insertions has not been well documented. Here, we assembled a Japanese genome using single-molecule real-time sequencing data and characterized insertions found in the assembled genome. We identified 3691 insertions ranging from 100?bps to ~10,000?bps in the assembled genome relative to the international reference sequence (GRCh38). To validate and characterize these insertions, we mapped short-reads from 1070 Japanese individuals and 728 individuals from eight other populations to insertions integrated into GRCh38. With…
In this PacBio User Group Meeting presentation, Tina Graves-Lindsay of the McDonnell Genome Institute and the Genome Reference Consortium speaks about the importance of phasing human reference genomes. Her team is now working on its fifteenth human genome assembly — part of a major effort to improve genomic representation of ethnic diversity — with a pipeline that generates 60-fold PacBio coverage for a de novo assembly, followed by scaffolding with other technologies. They are also using FALCON-Unzip to separate haplotypes, leading to reference-grade diploid assemblies. This approach has already helped resolve errors seen in other genomes and even the gold-standard…
In this presentation, Emily Hatas of PacBio offers a look a how SMRT Sequencing has changed over the years as well as the most common applications in human genome analysis: high-throughput structural variant detection; comprehensive variant detection; and de novo assembly of reference genomes.
PacBio customers discuss their applications of PacBio SMRT Sequencing and long reads, including Lemuel Racacho (Children’s Hospital of Eastern Ontario Research Institute), Matthew Blow (JGI), Yuta Suzuki (U. of Tokyo), Daniel Geraghty (Fred Hutchinson Cancer Center), and Mike Schatz (CSHL)
In his AGBT talk, Matthew Blow from the Joint Genome Institute describes high-throughput pipelines to annotate gene function and explore methylation in microbes. He uses transposon sequencing to annotate thousands of genes in bacteria and archaea. Later, he presents a study using SMRT Sequencing to generate complete methylomes for 232 prokaryotes, showing that orphan methylases appear to have a regulatory role.