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July 7, 2019

Smooth q-Gram, and its applications to detection of overlaps among long, error-prone sequencing reads

We propose smoothq-gram, the frst variant of q-gram that captures q-gram pair within a small edit distance. We apply smooth q-gram to the problem of detecting overlapping pairs of error-prone reads produced by single molecule real time sequencing (SMRT), which is the frst and most critical step of the de novo fragment assembly of SMRT reads. We have implemented and tested our algorithm on a set of real world benchmarks. Our empirical results demonstrated the signifcant superiority of our algorithm over the existing q-gram based algorithms in accuracy.


July 7, 2019

Sustaining global agriculture through rapid detection and deployment of genetic resistance to deadly crop diseases.

Contents Summary 45 I. Introduction 45 II. Targeted chromosome-based cloning via long-range assembly (TACCA) 46 III. Resistance gene cloning through mutational mapping (MutMap) 47 IV. Cloning through mutant chromosome sequencing (MutChromSeq) 47 V. Rapid cloning through resistance gene enrichment and sequencing (RenSeq) 49 VI. Cloning resistance genes through transcriptome profiling (RNAseq) 49 VII. Resistance gene deployment strategies 49 VIII. Conclusions 50 Acknowledgements 50 References 50 SUMMARY: Genetically encoded resistance is a major component of crop disease management. Historically, gene loci conferring resistance to pathogens have been identified through classical genetic methods. In recent years, accelerated gene cloning strategies have become available through advances in sequencing, gene capture and strategies for reducing genome complexity. Here, I describe these approaches with key emphasis on the isolation of resistance genes to the cereal crop diseases that are an ongoing threat to global food security. Rapid gene isolation enables their efficient deployment through marker-assisted selection and transgenic technology. Together with innovations in genome editing and progress in pathogen virulence studies, this creates further opportunities to engineer long-lasting resistance. These approaches will speed progress towards a future of farming using fewer pesticides.© 2017 Commonwealth of Australia. New Phytologist © 2017 New Phytologist Trust.


July 7, 2019

Draft genome assembly of the sheep scab mite, Psoroptes ovis.

Sheep scab, caused by infestation with Psoroptes ovis, is highly contagious, results in intense pruritus, and represents a major welfare and economic concern. Here, we report the first draft genome assembly and gene prediction of P. ovis based on PacBio de novo sequencing. The ~63.2-Mb genome encodes 12,041 protein-coding genes. Copyright © 2018 Burgess et al.


July 7, 2019

Satellite DNA evolution: old ideas, new approaches.

A substantial portion of the genomes of most multicellular eukaryotes consists of large arrays of tandemly repeated sequence, collectively called satellite DNA. The processes generating and maintaining different satellite DNA abundances across lineages are important to understand as satellites have been linked to chromosome mis-segregation, disease phenotypes, and reproductive isolation between species. While much theory has been developed to describe satellite evolution, empirical tests of these models have fallen short because of the challenges in assessing satellite repeat regions of the genome. Advances in computational tools and sequencing technologies now enable identification and quantification of satellite sequences genome-wide. Here, we describe some of these tools and how their applications are furthering our knowledge of satellite evolution and function. Copyright © 2018 Elsevier Ltd. All rights reserved.


July 7, 2019

Tigmint: correcting assembly errors using linked reads from large molecules.

Genome sequencing yields the sequence of many short snippets of DNA (reads) from a genome. Genome assembly attempts to reconstruct the original genome from which these reads were derived. This task is difficult due to gaps and errors in the sequencing data, repetitive sequence in the underlying genome, and heterozygosity. As a result, assembly errors are common. In the absence of a reference genome, these misassemblies may be identified by comparing the sequencing data to the assembly and looking for discrepancies between the two. Once identified, these misassemblies may be corrected, improving the quality of the assembled sequence. Although tools exist to identify and correct misassemblies using Illumina paired-end and mate-pair sequencing, no such tool yet exists that makes use of the long distance information of the large molecules provided by linked reads, such as those offered by the 10x Genomics Chromium platform. We have developed the tool Tigmint to address this gap.To demonstrate the effectiveness of Tigmint, we applied it to assemblies of a human genome using short reads assembled with ABySS 2.0 and other assemblers. Tigmint reduced the number of misassemblies identified by QUAST in the ABySS assembly by 216 (27%). While scaffolding with ARCS alone more than doubled the scaffold NGA50 of the assembly from 3 to 8 Mbp, the combination of Tigmint and ARCS improved the scaffold NGA50 of the assembly over five-fold to 16.4 Mbp. This notable improvement in contiguity highlights the utility of assembly correction in refining assemblies. We demonstrate the utility of Tigmint in correcting the assemblies of multiple tools, as well as in using Chromium reads to correct and scaffold assemblies of long single-molecule sequencing.Scaffolding an assembly that has been corrected with Tigmint yields a final assembly that is both more correct and substantially more contiguous than an assembly that has not been corrected. Using single-molecule sequencing in combination with linked reads enables a genome sequence assembly that achieves both a high sequence contiguity as well as high scaffold contiguity, a feat not currently achievable with either technology alone.


July 7, 2019

Correction of persistent errors in arabidopsis reference mitochondrial genomes.

Arabidopsis thaliana remains the foremost model system for plant genetics and genomics, and researchers rely on the accuracy of its genomic resources. The first completely sequenced angiosperm mitochondrial genome was obtained from Arabidopsis C24 (Unseld et al., 1997), and more recent efforts have produced additional Arabidopsis reference genomes, including one for Col-0, the most widely used ecotype in plant genetic research (Davila et al., 2011). These studies were based on older DNA sequencing methods, making them subject to errors associated with lower levels of sequencing coverage or the extremely short read lengths produced by early-generation Illumina technologies. Indeed, although the more recently published Arabidopsis mitochondrial reference genome sequences made substantial progress in improving upon earlier versions, they still have high error rates. By comparing publicly available Illumina sequence data to the Arabidopsis Col-0 reference genome, we found that it contains a sequence error every 2.4 kb on average, including 57 single-nucleotide polymorphisms (SNPs), 96 indels (up to 901 bp in size), and a large repeat-mediated rearrangement. Most of these errors appear to have been carried over from the original Arabidopsis mitochondrial genome sequence by reference-based assembly approaches, which has misled subsequent studies of plant mitochondrial mutation and molecular evolution by giving the false impression that the errors are naturally occurring variants present in multiple ecotypes. Building on the progress made by previous researchers, we provide a corrected reference sequence that we hope will serve as a useful community resource for future investigations in the field of plant mitochondrial genetics.


July 7, 2019

Next-generation sequencing of Haematococcus lacustris reveals an extremely large 1.35-megabase chloroplast genome.

Haematococcus lacustris is an industrially relevant microalga that is used for the production of the carotenoid astaxanthin. Here, we report the use of PacBio long-read sequencing to assemble the chloroplast genome of H. lacustris strain UTEX:2505. At 1.35?Mb, this is the largest assembled chloroplast of any plant or alga known to date. Copyright © 2018 Bauman et al.


July 7, 2019

A draft genome sequence for the Ixodes scapularis cell line, ISE6

Background: The tick cell line ISE6, derived from Ixodes scapularis, is commonly used for amplification and detection of arboviruses in environmental or clinical samples. Methods: To assist with sequence-based assays, we sequenced the ISE6 genome with single-molecule, long-read technology. Results: The draft assembly appears near complete based on gene content analysis, though it appears to lack some instances of repeats in this highly repetitive genome. The assembly appears to have separated the haplotypes at many loci. DNA short read pairs, used for validation only, mapped to the cell line assembly at a higher rate than they mapped to the Ixodes scapularis reference genome sequence. Conclusions: The assembly could be useful for filtering host genome sequence from sequence data obtained from cells infected with pathogens.


July 7, 2019

FusorSV: an algorithm for optimally combining data from multiple structural variation detection methods.

Comprehensive and accurate identification of structural variations (SVs) from next generation sequencing data remains a major challenge. We develop FusorSV, which uses a data mining approach to assess performance and merge callsets from an ensemble of SV-calling algorithms. It includes a fusion model built using analysis of 27 deep-coverage human genomes from the 1000 Genomes Project. We identify 843 novel SV calls that were not reported by the 1000 Genomes Project for these 27 samples. Experimental validation of a subset of these calls yields a validation rate of 86.7%. FusorSV is available at https://github.com/TheJacksonLaboratory/SVE .


July 7, 2019

Identification of repetitive DNA sequences in the Chrysanthemum boreale genome

We previously revealed that the Chrysanthemum boreale genome is highly repetitive; however, the types and nucleotide sequences of repetitive DNA in this diploid wild chrysanthemum are not known. Here, we characterized repetitive DNA sequences in the C. boreale genome by analysing genomic sequences obtained by Illumina sequencing and confirmed their repetitive nature by conducting fluorescence in situ hybridization (FISH) analyses. Annotation of the obtained DNA sequences revealed that microsatellite-containing genomic sequences exhibited similarity with genomic sequences in Chrysanthemum morifolium, indicating sequence conservation of repetitive DNA sequences between the two Chrysanthemum species. Two superfamilies of repetitive DNA, Copia and Gypsy, belonging to the long-terminal repeat (LTR) class of retrotransposons, are abundant in the C. boreale genome. We propose that Copia and Gypsy retroelements contribute to the current genome architecture of C. boreale. Whole genome sequencing, which is currently in progress, will reveal the extent to which these repetitive DNA sequences contribute.


July 7, 2019

The case for not masking away repetitive DNA

In the course of analyzing whole-genome data, it is common practice to mask or filter out repetitive regions of a genome, such as transposable elements and endogenous retroviruses, in order to focus only on genes and thus simplify the results. This Commentary is a plea from one member of the Mobile DNA community to all gene-centric researchers: please do not ignore the repetitive fraction of the genome. Please stop narrowing your findings by only analyzing a minority of the genome, and instead broaden your analyses to include the rich biology of repetitive and mobile DNA. In this article, I present four arguments supporting a case for retaining repetitive DNA in your genome-wide analysis.


July 7, 2019

Optimise wheat A-genome.

The wild einkorn wheat Triticum urartu (Tu) is the A-genome progenitor of tetraploid (AABB) and hexaploid (AABBDD) wheat. A draft genome of Tu was published in 2013, but a better reference sequence is urgently needed by scientists and breeders. Hong-Qing Ling, from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and colleagues have now completed a high-quality Tu genome using multiple methods.


July 7, 2019

ARKS: chromosome-scale scaffolding of human genome drafts with linked read kmers.

The long-range sequencing information captured by linked reads, such as those available from 10× Genomics (10xG), helps resolve genome sequence repeats, and yields accurate and contiguous draft genome assemblies. We introduce ARKS, an alignment-free linked read genome scaffolding methodology that uses linked reads to organize genome assemblies further into contiguous drafts. Our approach departs from other read alignment-dependent linked read scaffolders, including our own (ARCS), and uses a kmer-based mapping approach. The kmer mapping strategy has several advantages over read alignment methods, including better usability and faster processing, as it precludes the need for input sequence formatting and draft sequence assembly indexing. The reliance on kmers instead of read alignments for pairing sequences relaxes the workflow requirements, and drastically reduces the run time.Here, we show how linked reads, when used in conjunction with Hi-C data for scaffolding, improve a draft human genome assembly of PacBio long-read data five-fold (baseline vs. ARKS NG50?=?4.6 vs. 23.1 Mbp, respectively). We also demonstrate how the method provides further improvements of a megabase-scale Supernova human genome assembly (NG50?=?14.74 Mbp vs. 25.94 Mbp before and after ARKS), which itself exclusively uses linked read data for assembly, with an execution speed six to nine times faster than competitive linked read scaffolders (~?10.5 h compared to 75.7 h, on average). Following ARKS scaffolding of a human genome 10xG Supernova assembly (of cell line NA12878), fewer than 9 scaffolds cover each chromosome, except the largest (chromosome 1, n?=?13).ARKS uses a kmer mapping strategy instead of linked read alignments to record and associate the barcode information needed to order and orient draft assembly sequences. The simplified workflow, when compared to that of our initial implementation, ARCS, markedly improves run time performances on experimental human genome datasets. Furthermore, the novel distance estimator in ARKS utilizes barcoding information from linked reads to estimate gap sizes. It accomplishes this by modeling the relationship between known distances of a region within contigs and calculating associated Jaccard indices. ARKS has the potential to provide correct, chromosome-scale genome assemblies, promptly. We expect ARKS to have broad utility in helping refine draft genomes.


July 7, 2019

GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing.

Tandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations. We report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely – GtTR – which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation.We used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68 and 83% for capture sequence data and 200X WGS data respectively, improving to 87 and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25, 14, 12 and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results.The novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.


July 7, 2019

An improved approach for reconstructing consensus repeats from short sequence reads

Repeat elements are important components of most eukaryotic genomes. Most existing tools for repeat analysis rely either on high quality reference genomes or existing repeat libraries. Thus, it is still challenging to do repeat analysis for species with highly repetitive or complex genomes which often do not have good reference genomes or annotated repeat libraries. Recently we developed a computational method called REPdenovo that constructs consensus repeat sequences directly from short sequence reads, which outperforms an existing tool called RepARK. One major issue with REPdenovo is that it doesn’t perform well for repeats with relatively high divergence rates or low copy numbers. In this paper, we present an improved approach for constructing consensus repeats directly from short reads. Comparing with the original REPdenovo, the improved approach uses more repeat-related k-mers and improves repeat assembly quality using a consensus-based k-mer processing method.


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