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Auto Tag: Gene annotation

June 1, 2021  |  

Integrative biology of a fungus: Using PacBio SMRT Sequencing to interrogate the genome, epigenome, and transcriptome of Neurospora crassa.

PacBio SMRT Sequencing has the unique ability to directly detect base modifications in addition to the nucleotide sequence of DNA. Because eukaryotes use base modifications to regulate gene expression, the absence or presence of epigenetic events relative to the location of genes is critical to elucidate the function of the modification. Therefore an integrated approach that combines multiple omic-scale assays is necessary to study complex organisms. Here, we present an integrated analysis of three sequencing experiments: 1) DNA sequencing, 2) base-modification detection, and 3) Iso-seq analysis, in Neurospora crassa, a filamentous fungus that has been used to make many landmark discoveries in biochemistry and genetics. We show that de novo assembly of a new strain yields complete assemblies of entire chromosomes, and additionally contains entire centromeric sequences. Base-modification analyses reveal candidate sites of increased interpulse duration (IPD) ratio, that may signify regions of 5mC, 5hmC, or 6mA base modifications. Iso-seq method provides full-length transcript evidence for comprehensive gene annotation, as well as context to the base-modifications in the newly assembled genome. Projects that integrate multiple genome-wide assays could become common practice for identifying genomic elements and understanding their function in new strains and organisms.

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June 1, 2021  |  

Complex alternative splicing patterns in hematopoietic cell subpopulations revealed by third-generation long reads.

Background: Alternative splicing expands the repertoire of gene functions and is a signature for different cell populations. Here we characterize the transcriptome of human bone marrow subpopulations including progenitor cells to understand their contribution to homeostasis and pathological conditions such as atherosclerosis and tumor metastasis. To obtain full-length transcript structures, we utilized long reads in addition to RNA-seq for estimating isoform diversity and abundance. Method: Freshly harvested, viable human bone marrow tissues were extracted from discarded harvesting equipment and separated into total bone marrow (total), lineage-negative (lin-) progenitor cells and differentiated cells (lin+) by magnetic bead sorting with antibodies to surface markers of hematopoietic cell lineages. Sequencing was done with SOLiD, Illumina HiSeq (100bp paired-end reads), and PacBio RS II (full-length cDNA library protocol for 1 – 6 kb libraries). Short reads were assembled using both Trinity for de novo assembly and Cufflinks for genome-guided assembly. Full-length transcript consensus sequences were obtained for the PacBio data using the RS_IsoSeq protocol from PacBios SMRTAnalysis software. Quantitation for each sample was done independently for each sequencing platform using Sailfish to obtain the TPM (transcripts per million) using k-mer matching. Results: PacBios long read sequencing technology is capable of sequencing full-length transcripts up to 10 kb and reveals heretofore-unseen isoform diversity and complexity within the hematopoietic cell populations. A comparison of sequencing depth and de novo transcript assembly with short read, second-generation sequencing reveals that, while short reads provide precision in determining portions of isoform structure and supporting larger 5 and 3 UTR regions, it fails in providing a complete structure especially when multiple isoforms are present at the same locus. Increased breadth of isoform complexity is revealed by long reads that permits further elaboration of full isoform diversity and specific isoform abundance within each separate cell population. Sorting the distribution of major and minor isoforms reveals a cell population-specific balance focused on distinct genome loci and shows how tissue specificity and diversity are modulated by alternative splicing.

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June 1, 2021  |  

Rapid full-length Iso-Seq cDNA sequencing of rice mRNA to facilitate annotation and identify splice-site variation.

PacBio’s new Iso-Seq technology allows for rapid generation of full-length cDNA sequences without the need for assembly steps. The technology was tested on leaf mRNA from two model O. sativa ssp. indica cultivars – Minghui 63 and Zhenshan 97. Even though each transcriptome was not exhaustively sequenced, several thousand isoforms described genes over a wide size range, most of which are not present in any currently available FL cDNA collection. In addition, the lack of an assembly requirement provides direct and immediate access to complete mRNA sequences and rapid unraveling of biological novelties.

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June 1, 2021  |  

Genome analysis of a bacterium that causes lameness.

Lameness is a significant problem resulting in millions of dollars in lost revenue annually. In commercial broilers, the most common cause of lameness is bacterial chondronecrosis with osteomyelitis (BCO). We are using a wire flooring model to induce lameness attributable to BCO. We used 16S ribosomal DNA sequencing to determine that Staphylococcus spp. were the main species associated with BCO. Staphylococcus agnetis, which previously had not been isolated from poultry, was the principal species isolated from the majority of the bone lesion samples. Administering S. agnetis in the drinking water to broilers reared on wire flooring increased the incidence of BCO three-fold when compared with broilers drinking tap water (P = 0.001). We found that the minimum effective dose of Staphylococcus agnetis to induce BCO in broilers grown on wire flooring experiment is 105 cfu/ml. We used PacBio and Illumina sequencing to assemble a 2.4 Mbp contig representing the genome and a 34 kbp contig for the largest plasmid of S. agnetis. Annotation of this genome is underway through comparative genomics with other Staphylococcus genomes, and identification of virulence factors. Our goal is to elucidate genetic diversity, toxins, and pathogenicity determinants, for this poorly characterized species. Isolating pathogenic bacterial species, defining their likely route of transmission to broilers, and genomic analyses will contribute substantially to the development of measures for mitigating BCO losses in poultry.

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June 1, 2021  |  

Genome assembly strategies of the recent polyploid, Coffea arabica.

Arabica coffee, revered for its taste and aroma, has a complex genome. It is an allotetraploid (2n=4x=44) with a genome size of approximately 1.3 Gb, derived from the recent (< 0.6 Mya) hybridization of two diploid progenitors (2n=2x=22), C. canephora (710 Mb) and C. eugenioides (670 Mb). Both parental species diverged recently (< 4.2Mya) and their genomes are highly homologous. To facilitate assembly, a dihaploid plant was chosen for sequencing. Initial genome assembly attempts with short read data produced an assembly covering 1,031 Mb of the C. arabica genome with a contig L50 of 9kb. By implementation of long read PacBio at greater than 50x coverage and cutting-edge PacBio software, a de novo PacBio-only genome assembly was constructed that covers 1,042 Mb of the genome with an L50 of 267 kb. The two assemblies were assessed and compared to determine gene content, chimeric regions, and the ability to separate the parental genomes. A genetic map that contains 600 SSRs is being used for anchoring the contigs and improve the sub-genome differentiation together with the search of sub-genome specific SNPs. PacBio transcriptome sequencing is currently being added to finalize gene annotation of the polished assembly. The finished genome assembly will be used to guide re-sequencing assemblies of parental genomes (C. canephora and C. eugenioides) as well as a template for GBS analysis and whole genome re-sequencing of a set of C. arabica accessions representative of the species diversity. The obtained data will provide powerful genomic tools to enable more efficient coffee breeding strategies for this crop, which is highly susceptible to climate change and is the main source of income for millions of small farmers in producing countries.

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June 1, 2021  |  

A comprehensive lincRNA analysis: From conifers to trees

We have produced an updated annotation of the Norway spruce genome on the basis of an in siliconormalised set of RNA-Seq data obtained from 1,529 samples and comprising 15.5 billion paired-end Illumina HiSeq reads complemented by 18Mbp of PacBio cDNA data (3.2M sequences). In addition to augmenting and refining the previous protein coding gene annotation, here we focus on the addition of long intergenic non-coding RNA (lincRNA) and micro RNA (miRNA) genes. In addition to non-coding loci, our analyses also identified protein coding genes that had been missed by the initial genome annotation and enabled us to update the annotation of existing gene models. In particular, splice variant information, as supported by PacBio sequencing reads, has been added to the current annotation and previously fragmented gene models have been merged by scaffolding disjoint genomic scaffolds on the basis of transcript evidence. Using this refined annotation, a targeted analysis of the lincRNAs enabled their classification as i) deeply conserved, ii) conserved in seed plants iii) gymnosperm/conifer specific. Concurrently, complementary analyses were performed as part of the aspen genome project and the results of a comparative analysis of the lincRNAs conserved in both Norway spruce and Eurasian aspen enabled us to identify conserved and diverged expression profiles. At present, we are delving further into the expression results with the aim to functionally annotate the lincRNA genes, by developing a co-expression network analyses based GO annotation.

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February 5, 2021  |  

AGBT 2015 Highlights: Customer interviews day 1

PacBio customers discuss their applications of PacBio SMRT Sequencing and long reads, including Lemuel Racacho (Children’s Hospital of Eastern Ontario Research Institute), Matthew Blow (JGI), Yuta Suzuki (U. of Tokyo),…

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April 21, 2020  |  

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads.

The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective standalone technology for de novo assembly of human genomes. © 2019 John Wiley & Sons Ltd/University College London.

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