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July 19, 2019

SMRT Sequencing for parallel analysis of multiple targets and accurate SNP phasing.

Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously analyze one or a small number of genomic targets derived from multiple sources. In the budding yeast system, SMRT sequencing was used to analyze strand-exchange intermediates generated during mitotic recombination and to analyze genetic changes in a forward mutation assay. The general barcoding-SMRT approach was then extended to diffuse large B-cell lymphoma primary tumors and cell lines, where detected changes agreed with prior Illumina exome sequencing. A distinct advantage afforded by SMRT sequencing over other next-gen methods is that it immediately provides the linkage relationships between SNPs in the target segment sequenced. The strength of our approach for mutation/recombination studies (as well as linkage identification) derives from its inherent computational simplicity coupled with a lack of reliance on sophisticated statistical analyses. Copyright © 2015 Guo et al.

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July 19, 2019

The pineapple genome and the evolution of CAM photosynthesis.

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ? duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.

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July 19, 2019

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16?kilobases) reads with random errors, we assembled 99% (244?megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4?megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

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July 19, 2019

A supergene determines highly divergent male reproductive morphs in the ruff.

Three strikingly different alternative male mating morphs (aggressive ‘independents’, semicooperative ‘satellites’ and female-mimic ‘faeders’) coexist as a balanced polymorphism in the ruff, Philomachus pugnax, a lek-breeding wading bird. Major differences in body size, ornamentation, and aggressive and mating behaviors are inherited as an autosomal polymorphism. We show that development into satellites and faeders is determined by a supergene consisting of divergent alternative, dominant and non-recombining haplotypes of an inversion on chromosome 11, which contains 125 predicted genes. Independents are homozygous for the ancestral sequence. One breakpoint of the inversion disrupts the essential CENP-N gene (encoding centromere protein N), and pedigree analysis confirms the lethality of homozygosity for the inversion. We describe new differences in behavior, testis size and steroid metabolism among morphs and identify polymorphic genes within the inversion that are likely to contribute to the differences among morphs in reproductive traits.

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July 19, 2019

Lineage-specific methyltransferases define the methylome of the globally disseminated Escherichia coli ST131 clone.

Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone. Copyright © 2015 Forde et al.

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July 19, 2019

The power of Single Molecule Real-Time sequencing technology in the de novo assembly of a eukaryotic genome.

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. However, a large portion of the genomes still remains unassembled. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. The SMRT-based assembly produced 100 times longer contigs with 100 times smaller amount of gaps compared to the SGS-based assemblies. A detailed comparison between the assemblies revealed that the SMRT-based assembly enabled a more comprehensive gene annotation than the SGS-based assemblies where thousands of genes were missing or fragmented. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. We demonstrated that SMRT technology, though still needed support of SGS data, achieved a near-complete assembly of a eukaryotic genome.

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July 19, 2019

DNA methylation on N(6)-adenine in mammalian embryonic stem cells.

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N(6)-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N(6)-methyladenine. An increase of N(6)-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N(6)-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N(6)-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N(6)-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N(6)-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.

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July 19, 2019

Chromosomal-level assembly of the Asian seabass genome using long sequence reads and multi-layered scaffolding.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species’ native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

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July 19, 2019

Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing.

Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine resistance (R) gene sequence capture (RenSeq) with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone Rpi-amr3i. This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.

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July 19, 2019

Development and clinical application of an integrative genomic approach to personalized cancer therapy.

Personalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics.We developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data.We returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases. These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.

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July 19, 2019

Genomic changes following the reversal of a Y chromosome to an autosome in Drosophila pseudoobscura

Robertsonian translocations resulting in fusions between sex chromosomes and autosomes shape karyotype evolution by creating new sex chromosomes from autosomes. These translocations can also reverse sex chromosomes back into autosomes, which is especially intriguing given the dramatic differences between autosomes and sex chromosomes. To study the genomic events following a Y chromosome reversal, we investigated an autosome-Y translocation in Drosophila pseudoobscura. The ancestral Y chromosome fused to a small autosome (the dot chromosome) approximately 10–15 Mya. We used single molecule real-time sequencing reads to assemble the D. pseudoobscura dot chromosome, including this Y-to-dot translocation. We find that the intervening sequence between the ancestral Y and the rest of the dot chromosome is only ~78 Kb and is not repeat-dense, suggesting that the centromere now falls outside, rather than between, the fused chromosomes. The Y-to-dot region is 100 times smaller than the D. melanogaster Y chromosome, owing to changes in repeat landscape. However, we do not find a consistent reduction in intron sizes across the Y-to-dot region. Instead, deletions in intergenic regions and possibly a small ancestral Y chromosome size may explain the compact size of the Y-to-dot translocation.

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July 19, 2019

A comparison of tools for the simulation of genomic next-generation sequencing data.

Computer simulation of genomic data has become increasingly popular for assessing and validating biological models or for gaining an understanding of specific data sets. Several computational tools for the simulation of next-generation sequencing (NGS) data have been developed in recent years, which could be used to compare existing and new NGS analytical pipelines. Here we review 23 of these tools, highlighting their distinct functionality, requirements and potential applications. We also provide a decision tree for the informed selection of an appropriate NGS simulation tool for the specific question at hand.

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July 19, 2019

Analysis of tandem gene copies in maize chromosomal regions reconstructed from long sequence reads.

Haplotype variation not only involves SNPs but also insertions and deletions, in particular gene copy number variations. However, comparisons of individual genomes have been difficult because traditional sequencing methods give too short reads to unambiguously reconstruct chromosomal regions containing repetitive DNA sequences. An example of such a case is the protein gene family in maize that acts as a sink for reduced nitrogen in the seed. Previously, 41-48 gene copies of the alpha zein gene family that spread over six loci spanning between 30- and 500-kb chromosomal regions have been described in two Iowa Stiff Stalk (SS) inbreds. Analyses of those regions were possible because of overlapping BAC clones, generated by an expensive and labor-intensive approach. Here we used single-molecule real-time (Pacific Biosciences) shotgun sequencing to assemble the six chromosomal regions from the Non-Stiff Stalk maize inbred W22 from a single DNA sequence dataset. To validate the reconstructed regions, we developed an optical map (BioNano genome map; BioNano Genomics) of W22 and found agreement between the two datasets. Using the sequences of full-length cDNAs from W22, we found that the error rate of PacBio sequencing seemed to be less than 0.1% after autocorrection and assembly. Expressed genes, some with premature stop codons, are interspersed with nonexpressed genes, giving rise to genotype-specific expression differences. Alignment of these regions with those from the previous analyzed regions of SS lines exhibits in part dramatic differences between these two heterotic groups.

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July 19, 2019

Separate F-type plasmids have shaped the evolution of the H30 subclone of Escherichia coli sequence type 131.

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success.

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July 19, 2019

Emergence of a Homo sapiens-specific gene family and chromosome 16p11.2 CNV susceptibility.

Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates, including more recently the genomes of archaic hominins. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for approximately 1% of cases of autism and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. Here we reconstruct the evolutionary history of the locus and identify bolA family member 2 (BOLA2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95-kilobase-pair segment containing BOLA2 duplicated across the critical region approximately 282 thousand years ago (ka), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carried one or more copies of the duplication, which nearly fixed early in the human lineage-a pattern unlikely to have arisen so rapidly in the absence of selection (P?

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