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July 19, 2019

Long-read single molecule sequencing to resolve tandem gene copies: The Mst77Y region on the Drosophila melanogaster Y chromosome.

The autosomal gene Mst77F of Drosophila melanogaster is essential for male fertility. In 2010, Krsticevic et al. (Genetics 184: 295-307) found 18 Y-linked copies of Mst77F (“Mst77Y”), which collectively account for 20% of the functional Mst77F-like mRNA. The Mst77Y genes were severely misassembled in the then-available genome assembly and were identified by cloning and sequencing polymerase chain reaction products. The genomic structure of the Mst77Y region and the possible existence of additional copies remained unknown. The recent publication of two long-read assemblies of D. melanogaster prompted us to reinvestigate this challenging region of the Y chromosome. We found that the Illumina Synthetic Long Reads assembly failed in the Mst77Y region, most likely because of its tandem duplication structure. The PacBio MHAP assembly of the Mst77Y region seems to be very accurate, as revealed by comparisons with the previously found Mst77Y genes, a bacterial artificial chromosome sequence, and Illumina reads of the same strain. We found that the Mst77Y region spans 96 kb and originated from a 3.4-kb transposition from chromosome 3L to the Y chromosome, followed by tandem duplications inside the Y chromosome and invasion of transposable elements, which account for 48% of its length. Twelve of the 18 Mst77Y genes found in 2010 were confirmed in the PacBio assembly, the remaining six being polymerase chain reaction-induced artifacts. There are several identical copies of some Mst77Y genes, coincidentally bringing the total copy number to 18. Besides providing a detailed picture of the Mst77Y region, our results highlight the utility of PacBio technology in assembling difficult genomic regions such as tandemly repeated genes. Copyright © 2015 Krsticevic et al.

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July 19, 2019

DNA methylation on N6-adenine in C. elegans.

In mammalian cells, DNA methylation on the fifth position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC, as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N(6)-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylations of histone H3K4 and adenines and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 19, 2019

Single-Molecule Real-Time Sequencing combined with optical mapping yields completely finished fungal genome.

Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio-generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism’s biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes.Studying whole-genome sequences has become an important aspect of biological research. The advent of next-generation sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBio-generated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome analyses to facilitate functional studies into an organism’s biology. Copyright © 2015 Faino et al.

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July 19, 2019

Birth of a new gene on the Y chromosome of Drosophila melanogaster.

Contrary to the pattern seen in mammalian sex chromosomes, where most Y-linked genes have X-linked homologs, the Drosophila X and Y chromosomes appear to be unrelated. Most of the Y-linked genes have autosomal paralogs, so autosome-to-Y transposition must be the main source of Drosophila Y-linked genes. Here we show how these genes were acquired. We found a previously unidentified gene (flagrante delicto Y, FDY) that originated from a recent duplication of the autosomal gene vig2 to the Y chromosome of Drosophila melanogaster. Four contiguous genes were duplicated along with vig2, but they became pseudogenes through the accumulation of deletions and transposable element insertions, whereas FDY remained functional, acquired testis-specific expression, and now accounts for ~20% of the vig2-like mRNA in testis. FDY is absent in the closest relatives of D. melanogaster, and DNA sequence divergence indicates that the duplication to the Y chromosome occurred ~2 million years ago. Thus, FDY provides a snapshot of the early stages of the establishment of a Y-linked gene and demonstrates how the Drosophila Y has been accumulating autosomal genes.

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July 19, 2019

An improved genome reference for the African cichlid, Metriaclima zebra.

Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5× coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data.Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements.Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.

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July 19, 2019

Genetic variation and the de novo assembly of human genomes.

The discovery of genetic variation and the assembly of genome sequences are both inextricably linked to advances in DNA-sequencing technology. Short-read massively parallel sequencing has revolutionized our ability to discover genetic variation but is insufficient to generate high-quality genome assemblies or resolve most structural variation. Full resolution of variation is only guaranteed by complete de novo assembly of a genome. Here, we review approaches to genome assembly, the nature of gaps or missing sequences, and biases in the assembly process. We describe the challenges of generating a complete de novo genome assembly using current technologies and the impact that being able to perfectly sequence the genome would have on understanding human disease and evolution. Finally, we summarize recent technological advances that improve both contiguity and accuracy and emphasize the importance of complete de novo assembly as opposed to read mapping as the primary means to understanding the full range of human genetic variation.

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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

A diamond in the ruff.

Reference genomes are only as valuable as the scientific questions they can address. The ruff genome sequence papers exemplify three of the most important aspects of a useful genome: new biological insights, a high-quality resource and population variation data.

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July 19, 2019

Major improvements to the Heliconius melpomene genome assembly used to confirm 10 chromosome fusion events in 6 million years of butterfly evolution.

The Heliconius butterflies are a widely studied adaptive radiation of 46 species spread across Central and South America, several of which are known to hybridize in the wild. Here, we present a substantially improved assembly of the Heliconius melpomene genome, developed using novel methods that should be applicable to improving other genome assemblies produced using short read sequencing. First, we whole-genome-sequenced a pedigree to produce a linkage map incorporating 99% of the genome. Second, we incorporated haplotype scaffolds extensively to produce a more complete haploid version of the draft genome. Third, we incorporated ~20x coverage of Pacific Biosciences sequencing, and scaffolded the haploid genome using an assembly of this long-read sequence. These improvements result in a genome of 795 scaffolds, 275 Mb in length, with an N50 length of 2.1 Mb, an N50 number of 34, and with 99% of the genome placed, and 84% anchored on chromosomes. We use the new genome assembly to confirm that the Heliconius genome underwent 10 chromosome fusions since the split with its sister genus Eueides, over a period of about 6 million yr. Copyright © 2016 Davey et al.

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July 19, 2019

Long-read sequence assembly of the gorilla genome.

Accurate sequence and assembly of genomes is a critical first step for studies of genetic variation. We generated a high-quality assembly of the gorilla genome using single-molecule, real-time sequence technology and a string graph de novo assembly algorithm. The new assembly improves contiguity by two to three orders of magnitude with respect to previously released assemblies, recovering 87% of missing reference exons and incomplete gene models. Although regions of large, high-identity segmental duplications remain largely unresolved, this comprehensive assembly provides new biological insight into genetic diversity, structural variation, gene loss, and representation of repeat structures within the gorilla genome. The approach provides a path forward for the routine assembly of mammalian genomes at a level approaching that of the current quality of the human genome. Copyright © 2016, American Association for the Advancement of Science.

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July 19, 2019

Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes.

Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae. We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.

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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

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

AgIn: Measuring the landscape of CpG methylation of individual repetitive elements.

Determining the methylation state of regions with high copy numbers is challenging for second-generation sequencing, because the read length is insufficient to map reads uniquely, especially when repetitive regions are long and nearly identical to each other. Single-molecule real-time (SMRT) sequencing is a promising method for observing such regions, because it is not vulnerable to GC bias, it produces long read lengths, and its kinetic information is sensitive to DNA modifications.We propose a novel linear-time algorithm that combines the kinetic information for neighboring CpG sites and increases the confidence in identifying the methylation states of those sites. Using a practical read coverage of ~30-fold from an inbred strain medaka (Oryzias latipes), we observed that both the sensitivity and precision of our method on individual CpG sites were ~93.7%. We also observed a high correlation coefficient (R?=?0.884) between our method and bisulfite sequencing, and for 92.0% of CpG sites, methylation levels ranging over [0, 1] were in concordance within an acceptable difference 0.25. Using this method, we characterized the landscape of the methylation status of repetitive elements, such as LINEs, in the human genome, thereby revealing the strong correlation between CpG density and hypomethylation and detecting hypomethylation hot spots of LTRs and LINEs. We uncovered the methylation states for nearly identical active transposons, two novel LINE insertions of identity ~99% and length 6050 base pairs (bp) in the human genome, and 16 Tol2 elements of identity >99.8% and length 4682?bp in the medaka genome.AgIn (Aggregate on Intervals) is available at: https://github.com/hacone/AgIn CONTACT: ysuzuki@cb.k.u-tokyo.ac.jp, moris@cb.k.u-tokyo.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. © The Author(s) 2016. Published by Oxford University Press.

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July 19, 2019

Long read sequencing technology to solve complex genomic regions assembly in plants

Background: Numerous completed or on-going whole genome sequencing projects have highlighted the fact that obtaining a high quality genome sequence is necessary to address comparative genomics questions such as structural variations among genotypes and gain or loss of specific function. Despite the spectacular progress that has been made in sequencing technologies, obtaining accurate and reliable data is still a challenge, both at the whole genome scale and when targeting specific genomic regions. These problems are even more noticeable for complex plant genomes. Most plant genomes are known to be particularly challenging due to their size, high density of repetitive elements and various levels of ploidy. To overcome these problems, we have developed a strategy to reduce genome complexity by using the large insert BAC libraries combined with next generation sequencing technologies. Results: We compared two different technologies (Roche-454 and Pacific Biosciences PacBio RS II) to sequence pools of BAC clones in order to obtain the best quality sequence. We targeted nine BAC clones from different species (maize, wheat, strawberry, barley, sugarcane and sunflower) known to be complex in terms of sequence assembly. We sequenced the pools of the nine BAC clones with both technologies. We compared assembly results and highlighted differences due to the sequencing technologies used. Conclusions: We demonstrated that the long reads obtained with the PacBio RS II technology serve to obtain a better and more reliable assembly, notably by preventing errors due to duplicated or repetitive sequences in the same region.

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