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Auto Tag: Repeat structure

April 21, 2020  |  

Benchmarking Transposable Element Annotation Methods for Creation of a Streamlined, Comprehensive Pipeline

Sequencing technology and assembly algorithms have matured to the point that high-quality de novo assembly is possible for large, repetitive genomes. Current assemblies traverse transposable elements (TEs) and allow for annotation of TEs. There are numerous methods for each class of elements with unknown relative performance metrics. We benchmarked existing programs based on a curated library of rice TEs. Using the most robust programs, we created a comprehensive pipeline called Extensive de-novo TE Annotator (EDTA) that produces a condensed TE library for annotations of structurally intact and fragmented elements. EDTA is open-source and freely available: https://github.com/oushujun/EDTA.List of abbreviationsTETransposable ElementsLTRLong Terminal RepeatLINELong Interspersed Nuclear ElementSINEShort Interspersed Nuclear ElementMITEMiniature Inverted Transposable ElementTIRTerminal Inverted RepeatTSDTarget Site DuplicationTPTrue PositivesFPFalse PositivesTNTrue NegativeFNFalse NegativesGRFGeneric Repeat FinderEDTAExtensive de-novo TE Annotator

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April 21, 2020  |  

High satellite repeat turnover in great apes studied with short- and long-read technologies.

Satellite repeats are a structural component of centromeres and telomeres, and in some instances their divergence is known to drive speciation. Due to their highly repetitive nature, satellite sequences have been understudied and underrepresented in genome assemblies. To investigate their turnover in great apes, we studied satellite repeats of unit sizes up to 50?bp in human, chimpanzee, bonobo, gorilla, and Sumatran and Bornean orangutans, using unassembled short and long sequencing reads. The density of satellite repeats, as identified from accurate short reads (Illumina), varied greatly among great ape genomes. These were dominated by a handful of abundant repeated motifs, frequently shared among species, which formed two groups: (1) the (AATGG)n repeat (critical for heat shock response) and its derivatives; and (2) subtelomeric 32-mers involved in telomeric metabolism. Using the densities of abundant repeats, individuals could be classified into species. However clustering did not reproduce the accepted species phylogeny, suggesting rapid repeat evolution. Several abundant repeats were enriched in males vs. females; using Y chromosome assemblies or FIuorescent In Situ Hybridization, we validated their location on the Y. Finally, applying a novel computational tool, we identified many satellite repeats completely embedded within long Oxford Nanopore and Pacific Biosciences reads. Such repeats were up to 59?kb in length and consisted of perfect repeats interspersed with other similar sequences. Our results based on sequencing reads generated with three different technologies provide the first detailed characterization of great ape satellite repeats, and open new avenues for exploring their functions. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Centromeric Satellite DNAs: Hidden Sequence Variation in the Human Population.

The central goal of medical genomics is to understand the inherited basis of sequence variation that underlies human physiology, evolution, and disease. Functional association studies currently ignore millions of bases that span each centromeric region and acrocentric short arm. These regions are enriched in long arrays of tandem repeats, or satellite DNAs, that are known to vary extensively in copy number and repeat structure in the human population. Satellite sequence variation in the human genome is often so large that it is detected cytogenetically, yet due to the lack of a reference assembly and informatics tools to measure this variability, contemporary high-resolution disease association studies are unable to detect causal variants in these regions. Nevertheless, recently uncovered associations between satellite DNA variation and human disease support that these regions present a substantial and biologically important fraction of human sequence variation. Therefore, there is a pressing and unmet need to detect and incorporate this uncharacterized sequence variation into broad studies of human evolution and medical genomics. Here I discuss the current knowledge of satellite DNA variation in the human genome, focusing on centromeric satellites and their potential implications for disease.

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April 21, 2020  |  

Genomic and transcriptomic characterization of Pseudomonas aeruginosa small colony variants derived from a chronic infection model.

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA genes and suggest this may be the genetic switch for conversion to the SCV phenotype. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance because the appearance of SCVs during chronic infection is associated with declining lung function.

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April 21, 2020  |  

Complete chloroplast genome sequences of Kaempferia galanga and Kaempferia elegans: Molecular structures and comparative analysis.

Kaempferia galanga and Kaempferia elegans, which belong to the genus Kaempferia family Zingiberaceae, are used as valuable herbal medicine and ornamental plants, respectively. The chloroplast genomes have been used for molecular markers, species identification and phylogenetic studies. In this study, the complete chloroplast genome sequences of K. galanga and K. elegans are reported. Results show that the complete chloroplast genome of K. galanga is 163,811 bp long, having a quadripartite structure with large single copy (LSC) of 88,405 bp and a small single copy (SSC) of 15,812 bp separated by inverted repeats (IRs) of 29,797 bp. Similarly, the complete chloroplast genome of K. elegans is 163,555 bp long, having a quadripartite structure in which IRs of 29,773 bp length separates 88,020 bp of LSC and 15,989 bp of SSC. A total of 111 genes in K. galanga and 113 genes in K. elegans comprised 79 protein-coding genes and 4 ribosomal RNA (rRNA) genes, as well as 28 and 30 transfer RNA (tRNA) genes in K. galanga and K. elegans, respectively. The gene order, GC content and orientation of the two Kaempferia chloroplast genomes exhibited high similarity. The location and distribution of simple sequence repeats (SSRs) and long repeat sequences were determined. Eight highly variable regions between the two Kaempferia species were identified and 643 mutation events, including 536 single-nucleotide polymorphisms (SNPs) and 107 insertion/deletions (indels), were accurately located. Sequence divergences of the whole chloroplast genomes were calculated among related Zingiberaceae species. The phylogenetic analysis based on SNPs among eleven species strongly supported that K. galanga and K. elegans formed a cluster within Zingiberaceae. This study identified the unique characteristics of the entire K. galanga and K. elegans chloroplast genomes that contribute to our understanding of the chloroplast DNA evolution within Zingiberaceae species. It provides valuable information for phylogenetic analysis and species identification within genus Kaempferia.

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April 21, 2020  |  

Amplification-free long-read sequencing of TCF4 expanded trinucleotide repeats in Fuchs Endothelial Corneal Dystrophy.

Amplification of a CAG trinucleotide motif (CTG18.1) within the TCF4 gene has been strongly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Nevertheless, a small minority of clinically unaffected elderly patients who have expanded CTG18.1 sequences have been identified. To test the hypothesis that the CAG expansions in these patients are protected from FECD because they have interruptions within the CAG repeats, we utilized a combination of an amplification-free, long-read sequencing method and a new target-enrichment sequence analysis tool developed by Pacific Biosciences to interrogate the sequence structure of expanded repeats. The sequencing was successful in identifying a previously described interruption within an unexpanded allele and provided sequence data on expanded alleles greater than 2000 bases in length. The data revealed considerable heterogeneity in the size distribution of expanded repeats within each patient. Detailed analysis of the long sequence reads did not reveal any instances of interruptions to the expanded CAG repeats, but did reveal novel variants within the AGG repeats that flank the CAG repeats in two of the five samples from clinically unaffected patients with expansions. This first examination of the sequence structure of CAG repeats in CTG18.1 suggests that factors other than interruptions to the repeat structure account for the absence of disease in some elderly patients with repeat expansions in the TCF4 gene.

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April 21, 2020  |  

Characterizing the major structural variant alleles of the human genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity. Copyright © 2018 Elsevier Inc. All rights reserved.

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April 21, 2020  |  

Long-read sequencing identified intronic repeat expansions in SAMD12 from Chinese pedigrees affected with familial cortical myoclonic tremor with epilepsy.

The locus for familial cortical myoclonic tremor with epilepsy (FCMTE) has long been mapped to 8q24 in linkage studies, but the causative mutations remain unclear. Recently, expansions of intronic TTTCA and TTTTA repeat motifs within SAMD12 were found to be involved in the pathogenesis of FCMTE in Japanese pedigrees. We aim to identify the causative mutations of FCMTE in Chinese pedigrees.We performed genetic linkage analysis by microsatellite markers in a five-generation Chinese pedigree with 55 members. We also used array-comparative genomic hybridisation (CGH) and next-generation sequencing (NGS) technologies (whole-exome sequencing, capture region deep sequencing and whole-genome sequencing) to identify the causative mutations in the disease locus. Recently, we used low-coverage (~10×) long-read genome sequencing (LRS) on the PacBio Sequel and Oxford Nanopore platforms to identify the causative mutations, and used repeat-primed PCR for validation of the repeat expansions.Linkage analysis mapped the disease locus to 8q23.3-24.23. Array-CGH and NGS failed to identify causative mutations in this locus. LRS identified the intronic TTTCA and TTTTA repeat expansions in SAMD12 as the causative mutations, thus corroborating the recently published results in Japanese pedigrees.We identified the pentanucleotide repeat expansion in SAMD12 as the causative mutation in Chinese FCMTE pedigrees. Our study also suggested that LRS is an effective tool for molecular diagnosis of genetic disorders, especially for neurological diseases that cannot be positively diagnosed by conventional clinical microarray and NGS technologies. © Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.

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April 21, 2020  |  

Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome.

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5?kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the 'genome in a bottle' (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15?megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.

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April 21, 2020  |  

Tandem-genotypes: robust detection of tandem repeat expansions from long DNA reads.

Tandemly repeated DNA is highly mutable and causes at least 31 diseases, but it is hard to detect pathogenic repeat expansions genome-wide. Here, we report robust detection of human repeat expansions from careful alignments of long but error-prone (PacBio and nanopore) reads to a reference genome. Our method is robust to systematic sequencing errors, inexact repeats with fuzzy boundaries, and low sequencing coverage. By comparing to healthy controls, we prioritize pathogenic expansions within the top 10 out of 700,000 tandem repeats in whole genome sequencing data. This may help to elucidate the many genetic diseases whose causes remain unknown.

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April 21, 2020  |  

Comparative genomic and phylogenetic analyses of Populus section Leuce using complete chloroplast genome sequences

Species of Populus section Leuce are distributed throughout most parts of the Northern Hemisphere and have important economic and ecological significance. However, due to frequent hybridization within Leuce, the phylogenetic relationship between species has not been clarified. The chloroplast (cp) genome is characterized by maternal inheritance and relatively conservative mutation rates; thus, it is a powerful tool for building phylogenetic trees. In this study, we used the PacBio SEQUEL software to determine that the cp genome of Populus tomentosa has a length of 156,558 bp including a long single-copy region (84,717 bp), a small single-copy region (16,555 bp), and a pair of inverted repeat regions (27,643 bp). The cp genome contains 131 unique genes, including 37 transfer RNAs, 8 ribosomal RNAs, and 86 protein-coding genes. We compared the cp genomes of seven species of section Leuce and identified five cp DNA markers with >?1% variable sites. Phylogenetic analyses revealed two evolutionary branches for section Leuce. The species with the closest relationship with P. tomenstosa was P. adenopoda, followed by P. alba. These cp genome data will help to determine the cp evolution of section Leuce and further elucidate the origin of P. tomentosa.

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