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July 19, 2019

PacBio but not Illumina technology can achieve fast, accurate and complete closure of the high GC, complex Burkholderia pseudomallei two-chromosome genome

Although PacBio third-generation sequencers have improved the read lengths of genome sequencing which facilitates the assembly of complete genomes, no study has reported success in using PacBio data alone to completely sequence a two-chromosome bacterial genome from a single library in a single run. Previous studies using earlier versions of sequencing chemistries have at most been able to finish bacterial genomes containing only one chromosome with de novo assembly. In this study, we compared the robustness of PacBio RS II, using one SMRT cell and the latest P6-C4 chemistry, with Illumina HiSeq 1500 in sequencing the genome of Burkholderia pseudomallei, a bacterium which contains two large circular chromosomes, very high G+C content of 68–69%, highly repetitive regions and substantial genomic diversity, and represents one of the largest and most complex bacterial genomes sequenced, using a reference genome generated by hybrid assembly using PacBio and Illumina datasets with subsequent manual validation. Results showed that PacBio data with de novo assembly, but not Illumina, was able to completely sequence the B. pseudomallei genome without any gaps or mis-assemblies. The two large contigs of the PacBio assembly aligned unambiguously to the reference genome, sharing >99.9% nucleotide identities. Conversely, Illumina data assembled using three different assemblers resulted in fragmented assemblies (201–366 contigs), sharing only 92.2–100% and 92.0–100% nucleotide identities to chromosomes I and II reference sequences, respectively, with no indication that the B. pseudomallei genome consisted of two chromosomes with four copies of ribosomal operons. Among all assemblies, the PacBio assembly recovered the highest number of core and virulence proteins, and housekeeping genes based on whole-genome multilocus sequence typing (wgMLST). Most notably, assembly solely based on PacBio outperformed even hybrid assembly using both PacBio and Illumina datasets. Hybrid approach generated only 74 contigs, while the PacBio data alone with de novo assembly achieved complete closure of the two-chromosome B. pseudomallei genome without additional costly bench work and further sequencing. PacBio RS II using P6-C4 chemistry is highly robust and cost-effective and should be the platform of choice in sequencing bacterial genomes, particularly for those that are well-known to be difficult-to-sequence.

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July 19, 2019

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

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July 19, 2019

Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction.

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tapirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GH genes in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.IMPORTANCE The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus Caldicellulosiruptor rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain Caldicellulosiruptor species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in Caldicellulosiruptor bescii, the nuanced, substrate-specific in vivo roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in C. bescii, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats. We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntingtontextquoterights disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.

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July 19, 2019

The draft genome of tropical fruit durian (Durio zibethinus).

Durian (Durio zibethinus) is a Southeast Asian tropical plant known for its hefty, spine-covered fruit and sulfury and onion-like odor. Here we present a draft genome assembly of D. zibethinus, representing the third plant genus in the Malvales order and first in the Helicteroideae subfamily to be sequenced. Single-molecule sequencing and chromosome contact maps enabled assembly of the highly heterozygous durian genome at chromosome-scale resolution. Transcriptomic analysis showed upregulation of sulfur-, ethylene-, and lipid-related pathways in durian fruits. We observed paleopolyploidization events shared by durian and cotton and durian-specific gene expansions in MGL (methionine ?-lyase), associated with production of volatile sulfur compounds (VSCs). MGL and the ethylene-related gene ACS (aminocyclopropane-1-carboxylic acid synthase) were upregulated in fruits concomitantly with their downstream metabolites (VSCs and ethylene), suggesting a potential association between ethylene biosynthesis and methionine regeneration via the Yang cycle. The durian genome provides a resource for tropical fruit biology and agronomy.

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July 19, 2019

De novo PacBio long-read and phased avian genome assemblies correct and add to reference genes generated with intermediate and short reads.

Reference-quality genomes are expected to provide a resource for studying gene structure, function, and evolution. However, often genes of interest are not completely or accurately assembled, leading to unknown errors in analyses or additional cloning efforts for the correct sequences. A promising solution is long-read sequencing. Here we tested PacBio-based long-read sequencing and diploid assembly for potential improvements to the Sanger-based intermediate-read zebra finch reference and Illumina-based short-read Anna’s hummingbird reference, 2 vocal learning avian species widely studied in neuroscience and genomics. With DNA of the same individuals used to generate the reference genomes, we generated diploid assemblies with the FALCON-Unzip assembler, resulting in contigs with no gaps in the megabase range, representing 150-fold and 200-fold improvements over the current zebra finch and hummingbird references, respectively. These long-read and phased assemblies corrected and resolved what we discovered to be numerous misassemblies in the references, including missing sequences in gaps, erroneous sequences flanking gaps, base call errors in difficult-to-sequence regions, complex repeat structure errors, and allelic differences between the 2 haplotypes. These improvements were validated by single long-genome and transcriptome reads and resulted for the first time in completely resolved protein-coding genes widely studied in neuroscience and specialized in vocal learning species. These findings demonstrate the impact of long reads, sequencing of previously difficult-to-sequence regions, and phasing of haplotypes on generating the high-quality assemblies necessary for understanding gene structure, function, and evolution.© The Authors 2017. Published by Oxford University Press.

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July 19, 2019

The diversity, structure, and function of heritable adaptive immunity sequences in the Aedes aegypti genome.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions. Copyright © 2017 Elsevier Ltd. All rights reserved.

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July 19, 2019

Analysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline.

The Lyme disease spirochete evades the host immune system by combinatorial variation of VlsE, a surface antigen. Antigenic variation occurs via segmental gene conversion from contiguous silent cassettes into the vlsE locus. Because of the high degree of similarity between switch variants and the size of vlsE, short-read NGS technologies have been unsuitable for sequencing vlsE populations. Here we use PacBio sequencing technology coupled with the first fully-automated software pipeline (VAST) to accurately process NGS data by minimizing error frequency, eliminating heteroduplex errors and accurately aligning switch variants. We extend earlier studies by showing use of almost all of the vlsE SNP repertoire. In different tissues of the same mouse, 99.6% of the variants were unique, suggesting that dissemination of Borrelia burgdorferi is predominantly unidirectional with little tissue-to-tissue hematogenous dissemination. We also observed a similar number of variants in SCID and wild-type mice, a heatmap of location and frequency of amino acid changes on the 3D structure and note differences observed in SCID versus wild type mice that hint at possible amino acid function. Our observed selection against diversification of residues at the dimer interface in wild-type mice strongly suggests that dimerization is required for in vivo functionality of vlsE.© 2017 John Wiley & Sons Ltd.

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July 19, 2019

Comparative genomic analyses of Clavibacter michiganensis subsp. insidiosus and pathogenicity on Medicago truncatula.

Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,930 genes, with 401 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.

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July 19, 2019

Single molecule real-time (SMRT®) DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines.

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 IHIW cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, 2017-01-20), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n=10) and changes in zygosity (n=13), as well as previously unreported types (n=34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.This article is protected by copyright. All rights reserved.

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July 19, 2019

Centromere evolution and CpG methylation during vertebrate speciation.

Centromeres and large-scale structural variants evolve and contribute to genome diversity during vertebrate speciation. Here, we perform de novo long-read genome assembly of three inbred medaka strains that are derived from geographically isolated subpopulations and undergo speciation. Using single-molecule real-time (SMRT) sequencing, we obtain three chromosome-mapped genomes of length ~734, ~678, and ~744Mbp with a resource of twenty-two centromeric regions of length 20-345kbp. Centromeres are positionally conserved among the three strains and even between four pairs of chromosomes that were duplicated by the teleost-specific whole-genome duplication 320-350 million years ago. The centromeres do not all evolve at a similar pace; rather, centromeric monomers in non-acrocentric chromosomes evolve significantly faster than those in acrocentric chromosomes. Using methylation sensitive SMRT reads, we uncover centromeres are mostly hypermethylated but have hypomethylated sub-regions that acquire unique sequence compositions independently. These findings reveal the potential of non-acrocentric centromere evolution to contribute to speciation.

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July 19, 2019

Pacific Biosciences sequencing and IMGT/HighV-QUEST analysis of full-length single chain fragment variable from an in vivo selected phage-display combinatorial Library.

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires bothin vivoandin vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800?bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from anin vivoselection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of thein vivoselection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during thein vivopanning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767?bp, 53 of them covering the whole insert of 977?bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939?bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.

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July 19, 2019

Cytogenomic identification and long-read single molecule real-time (SMRT) sequencing of a Bardet-Biedl Syndrome 9 (BBS9) deletion.

Bardet-Biedl syndrome (BBS) is a recessive disorder characterized by heterogeneous clinical manifestations, including truncal obesity, rod-cone dystrophy, renal anomalies, postaxial polydactyly, and variable developmental delays. At least 20 genes have been implicated in BBS, and all are involved in primary cilia function. We report a 1-year-old male child from Guyana with obesity, postaxial polydactyly on his right foot, hypotonia, ophthalmologic abnormalities, and developmental delay, which together indicated a clinical diagnosis of BBS. Clinical chromosomal microarray (CMA) testing and high-throughput BBS gene panel sequencing detected a homozygous 7p14.3 deletion of exons 1-4 of BBS9 that was encompassed by a 17.5?Mb region of homozygosity at chromosome 7p14.2-p21.1. The precise breakpoints of the deletion were delineated to a 72.8?kb region in the proband and carrier parents by third-generation long-read single molecule real-time (SMRT) sequencing (Pacific Biosciences), which suggested non-homologous end joining as a likely mechanism of formation. Long-read SMRT sequencing of the deletion breakpoints also determined that the aberration included the neighboring RP9 gene implicated in retinitis pigmentosa; however, the clinical significance of this was considered uncertain given the paucity of reported cases with unambiguous RP9 mutations. Taken together, our study characterized a BBS9 deletion, and the identification of this shared haplotype in the parents suggests that this pathogenic aberration may be a BBS founder mutation in the Guyanese population. Importantly, this informative case also highlights the utility of long-read SMRT sequencing to map nucleotide breakpoints of clinically relevant structural variants.

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July 19, 2019

Sensitive detection of mitochondrial DNA variants for analysis of mitochondrial DNA-enriched extracts from frozen tumor tissue.

Large variation exists in mitochondrial DNA (mtDNA) not only between but also within individuals. Also in human cancer, tumor-specific mtDNA variation exists. In this work, we describe the comparison of four methods to extract mtDNA as pure as possible from frozen tumor tissue. Also, three state-of-the-art methods for sensitive detection of mtDNA variants were evaluated. The main aim was to develop a procedure to detect low-frequent single-nucleotide mtDNA-specific variants in frozen tumor tissue. We show that of the methods evaluated, DNA extracted from cytosol fractions following exonuclease treatment results in highest mtDNA yield and purity from frozen tumor tissue (270-fold mtDNA enrichment). Next, we demonstrate the sensitivity of detection of low-frequent single-nucleotide mtDNA variants (=1% allele frequency) in breast cancer cell lines MDA-MB-231 and MCF-7 by single-molecule real-time (SMRT) sequencing, UltraSEEK chemistry based mass spectrometry, and digital PCR. We also show de novo detection and allelic phasing of variants by SMRT sequencing. We conclude that our sensitive procedure to detect low-frequent single-nucleotide mtDNA variants from frozen tumor tissue is based on extraction of DNA from cytosol fractions followed by exonuclease treatment to obtain high mtDNA purity, and subsequent SMRT sequencing for (de novo) detection and allelic phasing of variants.

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July 19, 2019

A high-throughput approach for identification of nontuberculous mycobacteria in drinking water reveals relationship between water age and Mycobacterium avium.

Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTMrpoBgenes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age (i.e., combined distribution system residence time and home plumbing stagnation time) was associated with a greater relative abundance ofMycobacterium aviumsubsp.avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, includingMycobacterium abscessusandMycobacterium chelonaeOverall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM.IMPORTANCEAn extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTMrpoBgenes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). The extraction procedure recovered, on average, eight times as much NTM DNA and three times as much total DNA from DW as two widely used commercial DNA extraction kits. The combined DNA extraction and sequencing approach allowed high-throughput screening of DW samples to identify NTM, revealing that the relative abundance ofMycobacterium aviumsubsp.aviumincreased with water age. Furthermore, the two-step barcoding approach developed as part of the PacBio sequencing method makes this procedure highly adaptable, allowing it to be used for other target genes and species. Copyright © 2018 Haig et al.

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