As the foundation for scientific discoveries in genetic diversity, sequencing data must be accurate and complete. With highly accurate long-read sequencing, or HiFi sequencing, there is no longer a compromise between read length and accuracy. HiFi sequencing enables some of the highest quality de novo genome assemblies available today as well as comprehensive variant detection in human samples. PacBio HiFi libraries constructed using our standard library workflows require at least 3 µg of DNA input per 1 Gb of genome length, or ~10 µg for a human sample. For some samples it is not possible to extract this amount of…
PacBio bioinformatician Aaron Wenger presents this ASHG 2016 poster demonstrating human structural variation detection at varying coverage levels with SMRT Sequencing on the Sequel System. Results were compared to truth sets for well-characterized genomes. Results indicate that even low coverage of SMRT Sequencing makes it possible to detect hundreds of SVs that are missed in high-coverage short-read sequencing data.
This tutorial provides an overview of the Hierarchical Genome Assembly Process (HGAP4) de novo assembly analysis application. HGAP4 generates accurate de novo assemblies using only PacBio data. HGAP4 is suitable for assembling a wide range of genome sizes and complexity. HGAP4 now includes some support for diploid-aware assembly. This tutorial covers features of SMRT Link v5.0.0.
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…
Motivation: Third-generation sequencing technologies can sequence long reads, which is advancing the frontiers of genomics research. However, their high error rates prohibit accurate and efficient downstream analysis. This difficulty has motivated the development of many long read error correction tools, which tackle this problem through sampling redundancy and/or leveraging accurate short reads of the same biological samples. Existing studies to asses these tools use simulated data sets, and are not sufficiently comprehensive in the range of software covered or diversity of evaluation measures used. Results: In this paper, we present a categorization and review of long read error correction methods,…
Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both…
Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently…
Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens…
Acer yangbiense is a newly described critically endangered endemic maple tree confined to Yangbi County in Yunnan Province in Southwest China. It was included in a programme for rescuing the most threatened species in China, focusing on “plant species with extremely small populations (PSESP)”.We generated 64, 94, and 110 Gb of raw DNA sequences and obtained a chromosome-level genome assembly of A. yangbiense through a combination of Pacific Biosciences Single-molecule Real-time, Illumina HiSeq X, and Hi-C mapping, respectively. The final genome assembly is ~666 Mb, with 13 chromosomes covering ~97% of the genome and scaffold N50 sizes of 45 Mb.…
Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and…
Mycobacterium avium subsp. paratuberculosis is the causative agent of Johnetextquoterights disease (JD). Here, we report the complete genome sequence of Telford 9.2, a well-characterized representative strain of the M. avium subsp. paratuberculosis S subtype that is endemic in New Zealand and Australian sheep.
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 characterized 170 complete genome assemblies from clinical Bordetella pertussis isolates representing geographic and temporal diversity in the United States. These data capture genotypic shifts, including increased pertactin deficiency, occurring amid the current pertussis disease resurgence and provide a foundation for needed research to direct future public health control strategies.
In the past several years, single-molecule sequencing platforms, such as those by Pacific Biosciences and Oxford Nanopore Technologies, have become available to researchers and are currently being tested for clinical applications. They offer exceptionally long reads that permit direct sequencing through regions of the genome inaccessible or difficult to analyze by short-read platforms. This includes disease-causing long repetitive elements, extreme GC content regions, and complex gene loci. Similarly, these platforms enable structural variation characterization at previously unparalleled resolution and direct detection of epigenetic marks in native DNA. Here, we review how these technologies are opening up new clinical avenues that…
In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number…