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Friday, July 19, 2019

Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation.

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive…

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Friday, July 19, 2019

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated…

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Sunday, July 7, 2019

Dissecting a hidden gene duplication: the Arabidopsis thaliana SEC10 locus.

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment…

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Sunday, July 7, 2019

NOVOPlasty: de novo assembly of organelle genomes from whole genome data.

The evolution in next-generation sequencing (NGS) technology has led to the development of many different assembly algorithms, but few of them focus on assembling the organelle genomes. These genomes are used in phylogenetic studies, food identification and are the most deposited eukaryotic genomes in GenBank. Producing organelle genome assembly from whole genome sequencing (WGS) data would be the most accurate and least laborious approach, but a tool specifically designed for this task is lacking. We developed a seed-and-extend algorithm that assembles organelle genomes from whole genome sequencing (WGS) data, starting from a related or distant single seed sequence. The algorithm…

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Sunday, July 7, 2019

Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data.

The development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present.We present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and then performs read…

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Sunday, July 7, 2019

Cytosine methylation at CpCpG sites triggers accumulation of non-CpG methylation in gene bodies.

Methylation of cytosine is an epigenetic mark involved in the regulation of transcription, usually associated with transcriptional repression. In mammals, methylated cytosines are found predominantly in CpGs but in plants non-CpG methylation (in the CpHpG or CpHpH contexts, where H is A, C or T) is also present and is associated with the transcriptional silencing of transposable elements. In addition, CpG methylation is found in coding regions of active genes. In the absence of the demethylase of lysine 9 of histone 3 (IBM1), a subset of body-methylated genes acquires non-CpG methylation. This was shown to alter their expression and affect…

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Sunday, July 7, 2019

Improving and correcting the contiguity of long-read genome assemblies of three plant species using optical mapping and chromosome conformation capture data.

Long-read sequencing can overcome the weaknesses of short reads in the assembly of eukaryotic genomes, however, at present additional scaffolding is needed to achieve chromosome-level assemblies. We generated PacBio long-read data of the genomes of three relatives of the model plant Arabidopsis thaliana and assembled all three genomes into only a few hundred contigs. To improve the contiguities of these assemblies, we generated BioNano Genomics optical mapping and Dovetail Genomics chromosome conformation capture data for genome scaffolding. Despite their technical differences, optical mapping and chromosome conformation capture performed similarly and doubled N50 values. After improving both integration methods, assembly contiguity…

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Sunday, July 7, 2019

Patterns of polymorphism at the self-incompatibility locus in 1,083 Arabidopsis thaliana genomes.

Although the transition to selfing in the model plant Arabidopsis thaliana involved the loss of the self-incompatibility (SI) system, it clearly did not occur due to the fixation of a single inactivating mutation at the locus determining the specificities of SI (the S-locus). At least three groups of divergent haplotypes (haplogroups), corresponding to ancient functional S-alleles, have been maintained at this locus, and extensive functional studies have shown that all three carry distinct inactivating mutations. However, the historical process of loss of SI is not well understood, in particular its relation with the last glaciation. Here, we took advantage of…

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Sunday, July 7, 2019

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence.

During cell division, spindle fibers attach to chromosomes at centromeres. The DNA sequence at regional centromeres is fast evolving with no conserved genetic signature for centromere identity. Instead CENH3, a centromere-specific histone H3 variant, is the epigenetic signature that specifies centromere location across both plant and animal kingdoms. Paradoxically, CENH3 is also adaptively evolving. An ongoing question is whether CENH3 evolution is driven by a functional relationship with the underlying DNA sequence. Here, we demonstrate that despite extensive protein sequence divergence, CENH3 histones from distant species assemble centromeres on the same underlying DNA sequence. We first characterized the organization and…

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Sunday, July 7, 2019

Whole-genome restriction mapping by “subhaploid”-based RAD sequencing: An efficient and flexible approach for physical mapping and genome scaffolding.

Assembly of complex genomes using short reads remains a major challenge, which usually yields highly fragmented assemblies. Generation of ultradense linkage maps is promising for anchoring such assemblies, but traditional linkage mapping methods are hindered by the infrequency and unevenness of meiotic recombination that limit attainable map resolution. Here we develop a sequencing-based “in vitro” linkage mapping approach (called RadMap), where chromosome breakage and segregation are realized by generating hundreds of “subhaploid” fosmid/bacterial-artificial-chromosome clone pools, and by restriction site-associated DNA sequencing of these clone pools to produce an ultradense whole-genome restriction map to facilitate genome scaffolding. A bootstrap-based minimum spanning…

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Sunday, July 7, 2019

rHAT: fast alignment of noisy long reads with regional hashing.

Single Molecule Real-Time (SMRT) sequencing has been widely applied in cutting-edge genomic studies. However, it is still an expensive task to align the noisy long SMRT reads to reference genome by state-of-the-art aligners, which is becoming a bot-tleneck in applications with SMRT sequencing. Novel approach is on demand for improving the efficiency and effectiveness of SMRT read alignment.We propose Regional Hashing-based Alignment Tool (rHAT), a seed-and-extension-based read alignment approach specifically designed for noisy long reads. rHAT indexes reference genome by regional hash table (RHT), a hash table-based index which describes the short tokens within local windows of reference genome. In…

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Sunday, July 7, 2019

Exploring structural variants in environmentally sensitive gene families.

Environmentally sensitive plant gene families like NBS-LRRs, receptor kinases, defensins and others, are known to be highly variable. However, most existing strategies for discovering and describing structural variation in complex gene families provide incomplete and imperfect results. The move to de novo genome assemblies for multiple accessions or individuals within a species is enabling more comprehensive and accurate insights about gene family variation. Earlier array-based genome hybridization and sequence-based read mapping methods were limited by their reliance on a reference genome and by misplacement of paralogous sequences. Variant discovery based on de novo genome assemblies overcome the problems arising from…

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Sunday, July 7, 2019

1,135 genomes reveal the global pattern of polymorphism in Arabidopsis thaliana.

Arabidopsis thaliana serves as a model organism for the study of fundamental physiological, cellular, and molecular processes. It has also greatly advanced our understanding of intraspecific genome variation. We present a detailed map of variation in 1,135 high-quality re-sequenced natural inbred lines representing the native Eurasian and North African range and recently colonized North America. We identify relict populations that continue to inhabit ancestral habitats, primarily in the Iberian Peninsula. They have mixed with a lineage that has spread to northern latitudes from an unknown glacial refugium and is now found in a much broader spectrum of habitats. Insights into…

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Sunday, July 7, 2019

Sparc: a sparsity-based consensus algorithm for long erroneous sequencing reads.

Motivation. The third generation sequencing (3GS) technology generates long sequences of thousands of bases. However, its current error rates are estimated in the range of 15-40%, significantly higher than those of the prevalent next generation sequencing (NGS) technologies (less than 1%). Fundamental bioinformatics tasks such as de novo genome assembly and variant calling require high-quality sequences that need to be extracted from these long but erroneous 3GS sequences. Results. We describe a versatile and efficient linear complexity consensus algorithm Sparc to facilitate de novo genome assembly. Sparc builds a sparse k-mer graph using a collection of sequences from a targeted…

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Sunday, July 7, 2019

Recombination rate heterogeneity within Arabidopsis disease resistance genes.

Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and…

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