June 1, 2021  |  

Using whole exome sequencing and bacterial pathogen sequencing to investigate the genetic basis of pulmonary non-tuberculous mycobacterial infections.

Pulmonary non-tuberculous mycobacterial (PNTM) infections occur in patients with chronic lung disease, but also in a distinct group of elderly women without lung defects who share a common body morphology: tall and lean with scoliosis, pectus excavatum, and mitral valve prolapse. In order to characterize the human host susceptibility to PNTM, we performed whole exome sequencing (WES) of 44 individuals in extended families of patients with active PNTM as well as 55 additional unrelated individuals with PNTM. This unique collection of familial cohorts in PNTM represents an important opportunity for a high yield search for genes that regulate mucosal immunity. An average of 58 million 100bp paired-end Illumina reads per exome were generated and mapped to the hg19 reference genome. Following variant detection and classification, we identified 58,422 potentially high-impact SNPs, 97.3% of which were missense mutations. Segregating variants using the family pedigrees as well as comparisons to the unrelated individuals identified multiple potential variants associated with PNTM. Validations of these candidate variants in a larger PNTM cohort are underway. In addition to WES, we sequenced the genomes of 52 mycobacterial isolates, including 9 from these PNTM patients, to integrate host PNTM susceptibility with mycobacterial genotypes and gain insights into the key factors involved in this devastating disease. These genomes were sequenced using a combination of 454, Illumina, and PacBio platforms and assembled using multiple genome assemblers. The resulting genome sequences were used to identify mycobacterial genotypes associated with virulence, invasion, and drug resistance.


June 1, 2021  |  

Genome and transcriptome of the refeneration-competent flatworm, Macrostomum lignano

The free-living flatworm, Macrostomum lignano, much like its better known planarian relative, Schmidtea mediterranea, has an impressive regenerative capacity. Following injury, this species has the ability to regenerate almost an entirely new organism. This is attributable to the presence of an abundant somatic stem cell population, the neoblasts. These cells are also essential for the ongoing maintenance of most tissues, as their loss leads to irreversible degeneration of the animal. This set of unique properties makes a subset of flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of Macrostomum lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ~75% of its sequence being comprised of simple repeats and transposon sequences. This has made high quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130X coverage by long sequencing reads from the PacBio platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene expression patterns during regeneration, examining pathways important to stem cell function. As a whole, our data will provide a crucial resource for the community for the study not only of invertebrate evolution and phylogeny but also of regeneration and somatic pluripotency.


June 1, 2021  |  

Beyond Contiguity: Evaluating the accuracy of de novo genome assemblies

HiFi reads (>99% accurate, 15-20 kb) from the PacBio Sequel II System consistently provide complete and contiguous genome assemblies. In addition to completeness and contiguity, accuracy is of critical importance, as assembly errors complicate downstream analysis, particularly by disrupting gene frames. Metrics used to assess assembly accuracy include: 1) in-frame gene count, 2) kmer consistency, and 3) concordance to a benchmark, where discordances are interpreted as assembly errors. Genome in a Bottle (GIAB) provides a benchmark for the human genome with estimated accuracy of 99.9999% (Q60). Concordance for human HiFi assemblies exceeds Q50, which provides excellent genomes for downstream analysis, but presents a challenge that any new benchmark must significantly exceed Q50 or the discordance will represent the error rate of the benchmark. To establish benchmarks for Oryza sativa and Drosophila melanogaster, we collected draft references, Illumina short reads, and PacBio HiFi reads. By species, the benchmark was defined as regions of normal coverage that are not within 5 bp of a small variant or 50 bp of a structural variant. For both species, the benchmark regions span around 60% of the genome and HiFi assemblies achieve Q50 accuracy, which is notably more accurate than assemblies with other technologies and meets typical standards for a finished, reference-grade assembly. Here we present a protocol to generate benchmarks for any sample that rival the GIAB benchmark in accuracy. These benchmarks allow the comparison and improvement of genome assemblies and highlight the superior accuracy of assemblies generated with PacBio HiFi reads.


April 21, 2020  |  

The bracteatus pineapple genome and domestication of clonally propagated crops.

Domestication of clonally propagated crops such as pineapple from South America was hypothesized to be a ‘one-step operation’. We sequenced the genome of Ananas comosus var. bracteatus CB5 and assembled 513?Mb into 25 chromosomes with 29,412 genes. Comparison of the genomes of CB5, F153 and MD2 elucidated the genomic basis of fiber production, color formation, sugar accumulation and fruit maturation. We also resequenced 89 Ananas genomes. Cultivars ‘Smooth Cayenne’ and ‘Queen’ exhibited ancient and recent admixture, while ‘Singapore Spanish’ supported a one-step operation of domestication. We identified 25 selective sweeps, including a strong sweep containing a pair of tandemly duplicated bromelain inhibitors. Four candidate genes for self-incompatibility were linked in F153, but were not functional in self-compatible CB5. Our findings support the coexistence of sexual recombination and a one-step operation in the domestication of clonally propagated crops. This work guides the exploration of sexual and asexual domestication trajectories in other clonally propagated crops.


April 21, 2020  |  

Chromosome-level hybrid de novo genome assemblies as an attainable option for non-model organisms

The emergence of third generation sequencing (3GS; long-reads) is making closer the goal of chromosome-size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of non-model organisms. However, long-read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short-reads and long-reads provide an alternative efficient and cost-effective approach to generate de novo, chromosome-level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation is constantly being expanded and improved. This makes it difficult for non-experts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of non-model organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a non-model cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this non-model organism using the DBG2OLC pipeline.


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


April 21, 2020  |  

Variant Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize

Haplotype phasing of genetic variants is important for interpretation of the maize genome, population genetic analysis, and functional genomic analysis of allelic activity. Accordingly, accurate methods for phasing full-length isoforms are essential for functional genomics study. In this study, we performed an isoform-level phasing study in maize, using two inbred lines and their reciprocal crosses, based on single-molecule full-length cDNA sequencing. To phase and analyze full-length transcripts between hybrids and parents, we developed a tool called IsoPhase. Using this tool, we validated the majority of SNPs called against matching short read data and identified cases of allele-specific, gene-level, and isoform-level expression. Our results revealed that maize parental and hybrid lines exhibit different splicing activities. After phasing 6,847 genes in two reciprocal hybrids using embryo, endosperm and root tissues, we annotated the SNPs and identified large-effect genes. In addition, based on single-molecule sequencing, we identified parent-of-origin isoforms in maize hybrids, different novel isoforms between maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase power and accuracy in studies of allelic expression.


April 21, 2020  |  

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.


April 21, 2020  |  

deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index

Long-read RNA sequencing (RNA-seq) is promising to transcriptomics studies, however, the alignment of the reads is still a fundamental but non-trivial task due to the sequencing errors and complicated gene structures. We propose deSALT, a tailored two-pass long RNA-seq read alignment approach, which constructs graph-based alignment skeletons to sensitively infer exons, and use them to generate spliced reference sequence to produce refined alignments. deSALT addresses several difficult issues, such as small exons, serious sequencing errors and consensus spliced alignment. Benchmarks demonstrate that this approach has a better ability to produce high-quality full-length alignments, which has enormous potentials to transcriptomics studies.


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.


April 21, 2020  |  

A comprehensive evaluation of long read error correction methods

Motivation: Third-generation sequencing technologies can sequence long reads, which is advancing the frontiers of genomics research. However, their high error rates prohibit accurate and efficient downstream analysis. This difficulty has motivated the development of many long read error correction tools, which tackle this problem through sampling redundancy and/or leveraging accurate short reads of the same biological samples. Existing studies to asses these tools use simulated data sets, and are not sufficiently comprehensive in the range of software covered or diversity of evaluation measures used. Results: In this paper, we present a categorization and review of long read error correction methods, and provide a comprehensive evaluation of the corresponding long read error correction tools. Leveraging recent real sequencing data, we establish benchmark data sets and set up evaluation criteria for a comparative assessment which includes quality of error correction as well as run-time and memory usage. We study how trimming and long read sequencing depth affect error correction in terms of length distribution and genome coverage post-correction, and the impact of error correction performance on an important application of long reads, genome assembly. We provide guidelines for practitioners for choosing among the available error correction tools and identify directions for future research.


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