PacBio sequencing has been recognized as the gold-standard in microbial sequencing due to simultaneously providing long sequence reads (genome contiguity), high consensus accuracy (genome accuracy), minimal sequence bias (genome completeness),…
In this ASHG workshop presentation, Stuart Scott of the Icahn School of Medicine at Mount Sinai, presented on using the PacBio system for amplicon sequencing in pharmacogenomics and clinical genomics…
In this PacBio User Group Meeting presentation, Mount Sinai’s Ethan Ellis presents results from the HLS-CATCH method, which involves the use of the SageHLS instrument with CRISPR design methods to…
In this webinar we present Single Molecule, Real-Time (SMRT) Sequencing and the Iso-Seq method, which allow you to generate full-length cDNA sequences — no assembly required — to characterize transcript…
Understanding interactions among plants and the complex communities of organisms living on, in and around them requires more than one experimental approach. A new method for de novo metagenome assembly,…
Dr. Wenger gives attendees an update on PacBio’s long-read sequencing and variant detection capabilities on the Sequel II System and shares recommendations on how to design your own study using…
During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is <50% using these approaches, and there remain many rare genetic diseases with unknown cause. There may be many reasons for this, but one plausible explanation is that the responsible mutations are in regions of the genome that are difficult to sequence using conventional technologies (e.g., tandem-repeat expansion or complex chromosomal structural aberrations). Despite the drawbacks of high cost and a shortage of standard analytical methods, several studies have analyzed pathogenic changes in the genome using long-read sequencers. The results of these studies provide hope that further application of long-read sequencers to identify the causative mutations in unsolved genetic diseases may expand our understanding of the human genome and diseases. Such approaches may also be applied to molecular diagnosis and therapeutic strategies for patients with genetic diseases in the future.
Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing.Published by Elsevier Inc.
A major barrier to both metabolic engineering and fundamental biological studies is the lack of genetic tools in most microorganisms. One example is Clostridium thermocellum ATCC 27405T, where genetic tools are not available to help validate decades of hypotheses. A significant barrier to DNA transformation is restriction-modification systems, which defend against foreign DNA methylated differently than the host. To determine the active restriction-modification systems in this strain, we performed complete methylome analysis via single-molecule, real-time sequencing to detect 6-methyladenine and 4-methylcytosine and the rarely used whole-genome bisulfite sequencing to detect 5-methylcytosine. Multiple active systems were identified, and corresponding DNA methyltransferases were expressed from the Escherichia coli chromosome to mimic the C. thermocellum methylome. Plasmid methylation was experimentally validated and successfully electroporated into C. thermocellum ATCC 27405. This combined approach enabled genetic modification of the C. thermocellum-type strain and acts as a blueprint for transformation of other non-model microorganisms.
Horizontal gene transfer (HGT), the movement and genomic integration of DNA across species boundaries, is commonly associated with bacteria and other microorganisms, but functional HGT (fHGT) is increasingly being recognized in heterotrophic parasitic plants that obtain their nutrients and water from their host plants through direct haustorial feeding. Here, in the holoparasitic stem parasite Cuscuta, we identify 108?transcribed and probably functional HGT events in Cuscuta campestris and related species, plus 42?additional regions with host-derived transposon, pseudogene and non-coding sequences. Surprisingly, 18?Cuscuta fHGTs were acquired from the same gene families by independent HGT events in Orobanchaceae parasites, and the majority are highly expressed in the haustorial feeding structures in both lineages. Convergent retention and expression of HGT sequences suggests an adaptive role for specific additional genes in parasite biology. Between 16 and 20 of the transcribed HGT events are inferred as ancestral in Cuscuta based on transcriptome sequences from species across the phylogenetic range of the genus, implicating fHGT in the successful radiation of Cuscuta parasites. Genome sequencing of C. campestris supports transfer of genomic DNA-rather than retroprocessed RNA-as the mechanism of fHGT. Many of the C. campestris genes horizontally acquired are also frequent sources of 24-nucleotide small RNAs that are typically associated with RNA-directed DNA methylation. One HGT encoding a leucine-rich repeat protein kinase overlaps with a microRNA that has been shown to regulate host gene expression, suggesting that HGT-derived parasite small RNAs may function in the parasite-host interaction. This study enriches our understanding of HGT by describing a parasite-host system with unprecedented gene exchange that points to convergent evolution of HGT events and the functional importance of horizontally transferred coding and non-coding sequences.
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 with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the ~2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.
Haplotype phasing of genetic variants is important for interpretation of the maize genome, population genetic analysis, and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing full-length isoforms are essential for functional genomics study. In this study, we performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on single-molecule full-length cDNA sequencing. To phase and analyze full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short read data and identified cases of allele-specific, gene-level, and isoform-level expression. Our results revealed that maize parental and hybrid lines exhibit different splicing activities. After phasing 6,847 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, different novel isoforms between maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase power and accuracy in studies of allelic expression.
In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.
The antimicrobial resistance (AMR) crisis represents a serious threat to public health and has resulted in concentrated efforts to accelerate development of rapid molecular diagnostics for AMR. In combination with publicly-available web-based AMR databases, whole genome sequencing (WGS) offers the capacity for rapid detection of antibiotic resistance genes. Here we studied the concordance between WGS-based resistance prediction and phenotypic susceptibility testing results for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) clinical isolates using publicly-available tools and databases.Clinical isolates prospectively collected at the University of Pittsburgh Medical Center between December 2016 and December 2017 underwent WGS. Antibiotic resistance gene content was assessed from assembled genomes by BLASTn search of online databases. Concordance between WGS-predicted resistance profile and phenotypic susceptibility as well as sensitivity, specificity, positive and negative predictive values (NPV, PPV) were calculated for each antibiotic/organism combination, using the phenotypic results as the gold standard.Phenotypic susceptibility testing and WGS results were available for 1242 isolate/antibiotic combinations. Overall concordance was 99.3% with a sensitivity, specificity, PPV, NPV of 98.7% (95% CI, 97.2-99.5%), 99.6% (95 % CI, 98.8-99.9%), 99.3% (95% CI, 98.0-99.8%), 99.2% (95% CI, 98.3-99.7%), respectively. Additional identification of point mutations in housekeeping genes increased the concordance to 99.4% and the sensitivity to 99.3% (95% CI, 98.2-99.8%) and NPV to 99.4% (95% CI, 98.4-99.8%).WGS can be used as a reliable predicator of phenotypic resistance for both MRSA and VRE using readily-available online tools.Copyright © 2019. Published by Elsevier Ltd.
Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made towards understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57862 high-quality full-length mRNA sequences derived from 18362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length mRNA, we identified 4114 and 5880 pepper genes forming natural antisense transcript (NAT) genes in-cis and in-trans, respectively. Most of these genes accumulate small RNAs in their overlapping regions. By analyzing these NAT gene expression patterns in our transcriptome data, we identified many NAT pairs responsive to a variety of biological processes in pepper. Pepper formate dehydrogenase 1 (FDH1), which is required for R-gene-mediated disease resistance, may be regulated by nat-siRNAs and participate in a positive feedback loop in salicylic acid biosynthesis during resistance responses. Several cis-NAT pairs and subgroups of trans-NAT genes were responsive to pepper pericarp and placenta development, which may play roles in capsanthin and capsaicin biosynthesis. Using a comparative genomics approach, the evolutionary mechanisms of cis-NATs were investigated, and we found that an increase in intergenic sequences accounted for the loss of most cis-NATs, while transposon insertion contributed to the formation of most new cis-NATs. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.
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