A major contributor to inter-individual genomic variability is copy number variation (CNV). CNVs change the diploid status of the DNA, involve one or multiple genes, and may disrupt coding regions, affect regulatory elements, or change gene dosage. While some of these changes may have no phenotypic consequences, others underlie disease, explain evolutionary processes, or impact the response to medication.
Eliminating errors in next-generation DNA sequencing has proved challenging. Here we present error-correction code (ECC) sequencing, a method to greatly improve sequencing accuracy by combining fluorogenic sequencing-by-synthesis (SBS) with an information theory-based error-correction algorithm. ECC embeds redundancy in sequencing reads by creating three orthogonal degenerate sequences, generated by alternate dual-base reactions. This is similar to encoding and decoding strategies that have proved effective in detecting and correcting errors in information communication and storage. We show that, when combined with a fluorogenic SBS chemistry with raw accuracy of 98.1%, ECC sequencing provides single-end, error-free sequences up to 200 bp. ECC approaches should enable accurate identification of extremely rare genomic variations in various applications in biology and medicine.
DNA N6-methyladenine (6mA) is newly rediscovered as a potential epigenetic mark across a more diverse range of eukaryotes than previously realized. As a unicellular model organism, Tetrahymena thermophila is among the first eukaryotes reported to contain 6mA modification. However, lack of comprehensive information about 6mA distribution hinders further investigations into its function and regulatory mechanism. In this study, we provide the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing. We provide evidence that 6mA occurs mostly in the AT motif of the linker DNA regions. More strikingly, these linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes and/or H2A.Z-containing nucleosomes. We also find that 6mA is exclusively associated with RNA polymerase II (Pol II)-transcribed genes, but is not an unambiguous mark for active transcription. These results support that 6mA is an integral part of the chromatin landscape shaped by adenosine triphosphate (ATP)-dependent chromatin remodeling and transcription.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
The order Hymenochaetales of white rot fungi contain some of the most aggressive wood decayers causing tree deaths around the world. Despite their ecological importance and the impact of diseases they cause, little is known about the evolution and transmission patterns of these pathogens. Here, we sequenced and undertook comparative genomic analyses of Hymenochaetales genomes using brown root rot fungus Phellinus noxius, wood-decomposing fungus Phellinus lamaensis, laminated root rot fungus Phellinus sulphurascens and trunk pathogen Porodaedalea pini. Many gene families of lignin-degrading enzymes were identified from these fungi, reflecting their ability as white rot fungi. Comparing against distant fungi highlighted the expansion of 1,3-beta-glucan synthases in P. noxius, which may account for its fast-growing attribute. We identified 13 linkage groups conserved within Agaricomycetes, suggesting the evolution of stable karyotypes. We determined that P. noxius has a bipolar heterothallic mating system, with unusual highly expanded ~60 kb A locus as a result of accumulating gene transposition. We investigated the population genomics of 60 P. noxius isolates across multiple islands of the Asia Pacific region. Whole-genome sequencing showed this multinucleate species contains abundant poly-allelic single nucleotide polymorphisms with atypical allele frequencies. Different patterns of intra-isolate polymorphism reflect mono-/heterokaryotic states which are both prevalent in nature. We have shown two genetically separated lineages with one spanning across many islands despite the geographical barriers. Both populations possess extraordinary genetic diversity and show contrasting evolutionary scenarios. These results provide a framework to further investigate the genetic basis underlying the fitness and virulence of white rot fungi.© 2017 John Wiley & Sons Ltd.
HLA-B*08:183 differs from HLA-B*08:01:01 by a single substitution in exon 5.© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Raphanus sativus L. includes an annual root vegetable crop, radish, and diverse wild species. R. sativus has a long history of domestication, but its phylogenetic position in the tribe Brassiceae is controversial. A comprehensive analysis of the R. sativus genome will provide fundamental information about the structure of its genome, evolutionary features of polyploidy, and significant insight for phylogenetic delimitation of this species. Diverse genomic resources, including a high-density genetic map, clone libraries, cytogenetic data, and transcriptome data, have been developed to sequence the genome. Recently, the R. sativus cv. ‘WK10039’ (2n = 18, 510.8 Mb) genome was sequenced and assembled into nine chromosome pseudomolecules spanning >98% of the gene space. Comparative mapping of the tPCK-like ancestral genome based on conserved ortholog set markers and proteome comparison revealed that the R. sativus genome has intermediate characteristics between the Brassica A/C and B genomes with triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between R. sativus and diploid Brassica species provide genomic evidence for species delimitation of R. sativus and reconstruction of the mesohexaploid ancestral genome.
Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.
The free-living flatworm Macrostomum lignano is a hidden tetraploid. Its genome was formed by a recent whole genome duplication followed by chromosome fusions. Its karyotype (2n = 8) consists of a pair of large chromosomes (MLI1), which contain regions of all other chromosomes, and three pairs of small metacentric chromosomes. Comparison of MLI1 with metacentrics was performed by painting with microdissected DNA probes and fluorescent in situ hybridization of unique DNA fragments. Regions of MLI1 homologous to small metacentrics appeared to be contiguous. Besides the loss of DNA repeat clusters (pericentromeric and telomeric repeats and the 5S rDNA cluster) from MLI1, the difference between small metacentrics MLI2 and MLI4 and regions homologous to them in MLI1 were revealed. Abnormal karyotypes found in the inbred DV1/10 subline were analyzed, and structurally rearranged chromosomes were described with the painting technique, suggesting the mechanism of their origin. The revealed chromosomal rearrangements generate additional diversity, opening the way toward massive loss of duplicated genes from a duplicated genome. Our findings suggest that the karyotype of M. lignano is in the early stage of genome diploidization after whole genome duplication, and further studies on M. lignano and closely related species can address many questions about karyotype evolution in animals.
Structural Variation (SV) detection from short-read whole genome sequencing is error prone, presenting significant challenges for population or family-based studies of disease.Here we describe SV2, a machine-learning algorithm for genotyping deletions and duplications from paired-end sequencing data. SV2 can rapidly integrate variant calls from multiple structural variant discovery algorithms into a unified call set with high genotyping accuracy and capability to detect de novo mutations. SV2 is freely available on GitHub (https://github.com/dantaki/SV2).Supplementary data are available at Bioinformatics online.© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
In July 2014, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O55:H7 in England involved 31 patients, 13 (42%) of whom had hemolytic uremic syndrome. Isolates were sequenced, and the sequences were compared with publicly available sequences of E. coli O55:H7 and O157:H7. A core-genome phylogeny of the evolutionary history of the STEC O55:H7 outbreak strain revealed that the most parsimonious model was a progenitor enteropathogenic O55:H7 sorbitol-fermenting strain, lysogenized by a Shiga toxin (Stx) 2a-encoding phage, followed by loss of the ability to ferment sorbitol because of a non-sense mutation in srlA. The parallel, convergent evolutionary histories of STEC O157:H7 and STEC O55:H7 may indicate a common driver in the evolutionary process. Because emergence of STEC O157:H7 as a clinically significant pathogen was associated with acquisition of the Stx2a-encoding phage, the emergence of STEC O55:H7 harboring the stx2a gene is of public health concern.
Next-generation sequencing and traditional Sanger sequencing methods are of great significance in unraveling the complexity of plant genomes. These are constantly generating heaps of sequence data to be analyzed, annotated and stored. This has created a revolutionary demand for bioinformatics tools and software that can perform these functions. A large number of potentially useful bioinformatics tools and plant genome databases are created that have greatly simplified the analysis and storage of vast amounts of sequence data. The information garnered using the available bioinformatics methods have greatly helped in understanding the plant genome structure. Despite the availability of a good number of such tools, the information pouring from single gene-sequencing, and various whole-genome sequencing projects is overwhelming; thus, further innovations and improved methods are needed to sift through this sequence data, and assemble genomes. The current review focuses on diverse bioinformatics approaches and methods developed to systematically analyze and store plant sequence data. Finally, it outlines the bottlenecks in plant genome analysis, and some possible solutions that could be utilized to overcome the problems associated with plant genome analysis.
Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10-8and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs. Copyright © 2017 the Author(s). Published by PNAS.
X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease associated with an antisense insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within an intron ofTAF1This unique insertion coincides with six additional noncoding sequence changes inTAF1, the gene that encodes TATA-binding protein-associated factor-1, which appear to be inherited together as an identical haplotype in all reported cases. Here we examined the sequence of this SVA in XDP patients (n= 140) and detected polymorphic variation in the length of a hexanucleotide repeat domain, (CCCTCT)nThe number of repeats in these cases ranged from 35 to 52 and showed a highly significant inverse correlation with age at disease onset. Because other SVAs exhibit intrinsic promoter activity that depends in part on the hexameric domain, we assayed the transcriptional regulatory effects of varying hexameric lengths found in the unique XDP SVA retrotransposon using luciferase reporter constructs. When inserted sense or antisense to the luciferase reading frame, the XDP variants repressed or enhanced transcription, respectively, to an extent that appeared to vary with length of the hexamer. Further in silico analysis of this SVA sequence revealed multiple motifs predicted to form G-quadruplexes, with the greatest potential detected for the hexameric repeat domain. These data directly link sequence variation within the XDP-specific SVA sequence to phenotypic variability in clinical disease manifestation and provide insight into potential mechanisms by which this intronic retroelement may induce transcriptional interference inTAF1expression. Copyright © 2017 the Author(s). Published by PNAS.
Proximate environmental effects on metamorphosis have been explored in many vertebrate systems, but less attention has been devoted to how the environment affects developmental morphological change in mammals. Understanding proximate environmental effects on mammalian morphological change, particularly changes involving skin replacement, may aid in the design of therapeutic strategies to address severe burn or other debilitating injuries. Here, we specifically explore effects of celerity broadly, and we present results showing rapid change in mammalian morphological development following encountering maximum celerity. Morphological changes were pronounced within 96 hours and included at least partial regeneration of skin and organs as well as an elevated somatic mutation rate. Significantly, this high mutation rate did not result in detectable loss of fertility or viability of offspring. Overall, our findings strongly suggest that extreme celerity, an environmental factor rarely considered, can produce strikingly rapid developmental changes in morphology even in mammalian systems and open the door to future studies on the impact of celerity on genetics and morphology.
Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted “noncoding RNAs” to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.© 2017 Prasad et al.; Published by Cold Spring Harbor Laboratory Press.
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