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Auto Tag: Eukaryotic

June 1, 2021  |  

Cogent: Reconstructing the coding genome from full-length transcriptome sequences

For highly complex and large genomes, a well-annotated genome may be computationally challenging and costly, yet the study of alternative splicing events and gene annotations usually rely on the existence of a genome. Long-read sequencing technology provides new opportunities to sequence full-length cDNAs, avoiding computational challenges that short read transcript assembly brings. The use of single molecule, real-time sequencing from Pacific Biosciences to sequence transcriptomes (the Iso-SeqTM method), which produces de novo, high-quality, full-length transcripts, has revealed an astonishing amount of alternative splicing in eukaryotic species. With the Iso-Seq method, it is now possible to reconstruct the transcribed regions of the genome using just the transcripts themselves. We present Cogent, a tool for finding gene families and reconstructing the coding genome in the absence of a reference genome. Cogent uses k-mer similarities to first partition the transcripts into different gene families. Then, for each gene family, the transcripts are used to build a splice graph. Cogent identifies bubbles resulting from sequencing errors, minor variants, and exon skipping events, and attempts to resolve each splice graph down to the minimal set of reconstructed contigs. We apply Cogent to a Cuttlefish Iso-Seq dataset, for which there is a highly fragmented, Illumina-based draft genome assembly and little annotation. We show that Cogent successfully discovers gene families and can reconstruct the coding region of gene loci. The reconstructed contigs can then be used to visualize alternative splicing events, identify minor variants, and even be used to improve genome assemblies.

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June 1, 2021  |  

Full-length cDNA sequencing for genome annotation and analysis of alternative splicing

In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties and functions. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be understated. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for gene discovery and annotation. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 – 10 kb, fully intact RNA molecules can be sequenced using SMRT Sequencing without requiring fragmentation or post-sequencing assembly. Our open-source computational pipeline delivers high-quality, non-redundant sequences for unambiguous identification of alternative splicing events, alternative transcriptional start sites, polyA tail, and gene fusion events. We applied the Iso-Seq method to the maize (Zea mays) inbred line B73. Full-length cDNAs from six diverse tissues were barcoded and sequenced across multiple size-fractionated SMRTbell libraries. A total of 111,151 unique transcripts were identified. More than half of these transcripts (57%) represented novel, sometimes tissue-specific, isoforms of known genes. In addition to the 2250 novel coding genes and 860 lncRNAs discovered, the Iso-Seq dataset corrected errors in existing gene models, highlighting the value of full-length transcripts for whole gene annotations.

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June 1, 2021  |  

A comprehensive study of the sugar pine (Pinus lambertiana) transcriptome implemented through diverse next-generation sequencing approaches

The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46 SMRT cells) reads, 33,720 unigenes were de novo assembled. Comparison of sequencing technologies revealed improved coverage with Illumina HiSeq reads and better splice variant detection with PacBio Iso-Seq reads. The genes identified as unique to each library ranges from 199 transcripts (basket seedling) to 3,482 transcripts (female cones). In total, 10,026 transcripts were shared by all libraries. Genes differentially expressed in response to these provided insight on abiotic and biotic stress responses. To analyze orthologous sequences, we compared the translated sequences against 19 plant species, identifying 7,229 transcripts that clustered uniquely among the conifers. We have generated here a high quality transcriptome from one WPBR susceptible and one WPBR resistant sugar pine individual. Through the comprehensive tissue sampling and the depth of the sequencing achieved, detailed information on disease resistance can be further examined.

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June 1, 2021  |  

From Sequencing to Chromosomes: New de novo assembly and scaffolding methods improve the goat reference genome

Single-molecule sequencing is now routinely used to assemble complete, high-quality microbial genomes, but these assembly methods have not scaled well to large genomes. To address this problem, we previously introduced the MinHash Alignment Process (MHAP) for overlapping single-molecule reads using probabilistic, locality-sensitive hashing. Integrating MHAP with Celera Assembler (CA) has enabled reference-grade assemblies of model organisms, revealing novel heterochromatic sequences and filling low-complexity gap sequences in the GRCh38 human reference genome. We have applied our methods to assemble the San Clemente goat genome. Combining single-molecule sequencing from Pacific Biosciences and BioNano Genomics generates and assembly that is over 150-fold more contiguous than the latest Capra hircus reference. In combination with Hi-C sequencing, the assembly surpasses reference assemblies, de novo, with minimal manual intervention. The autosomes are each assembled into a single scaffold. Our assembly provides a more complete gene reconstruction, better alignments with Goat 52k chip, and improved allosome reconstruction. In addition to providing increased continuity of sequence, our assembly achieves a higher BUSCO completion score (84%) than the existing goat reference assembly suggesting better quality annotation of gene models. Our results demonstrate that single-molecule sequencing can produce near-complete eukaryotic genomes at modest cost and minimal manual effort.

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June 1, 2021  |  

Reconstruction of the spinach coding genome using full-length transcriptome without a reference genome

For highly complex and large genomes, a well-annotated genome may be computationally challenging and costly, yet the study of alternative splicing events and gene annotations usually rely on the existence of a genome. Long-read sequencing technology provides new opportunities to sequence full-length cDNAs, avoiding computational challenges that short read transcript assembly brings. The use of single molecule, real-time sequencing from PacBio to sequence transcriptomes (the Iso-Seq method), which produces de novo, high-quality, full-length transcripts, has revealed an astonishing amount of alternative splicing in eukaryotic species. With the Iso-Seq method, it is now possible to reconstruct the transcribed regions of the genome using just the transcripts themselves. We present Cogent, a tool for finding gene families and reconstructing the coding genome in the absence of a high-quality reference genome. Cogent uses k-mer similarities to first partition the transcripts into different gene families. Then, for each gene family, the transcripts are used to build a splice graph. Cogent identifies bubbles resulting from sequencing errors, minor variants, and exon skipping events, and attempts to resolve each splice graph down to the minimal set of reconstructed contigs. We apply Cogent to the Iso-Seq data for spinach, Spinacia oleracea, for which there is also a PacBio-based draft genome to validate the reconstruction. The Iso-Seq dataset consists of 68,263 fulllength, Quiver-polished transcript sequences ranging from 528 bp to 6 kbp long (mean: 2.1 kbp). Using the genome mapping as ground truth, we found that 95% (8045/8446) of the Cogent gene families found corresponded to a single genomic loci. For families that contained multiple loci, they were often homologous genes that would be categorized as belonging to the same gene family. Coding genome reconstruction was then performed individually for each gene family. A total of 86% (7283/8446) of the gene families were resolved to a single contig by Cogent, and was validated to be also a single contig in the genome. In 59 cases, Cogent reconstructed a single contig, however the contig corresponded to 2 or more loci in the genome, suggesting possible scaffolding opportunities. In 24 cases, the transcripts had no hits to the genome, though Pfam and BLAST searches of the transcripts show that they were indeed coding, suggesting that the genome is missing certain coding portions. Given the high quality of the spinach genome, we were not surprised to find that Cogent only minorly improved the genome space. However the ability of Cogent to accurately identify gene families and reconstruct the coding genome in a de novo fashion shows that it will be extremely powerful when applied to datasets for which there is no or low-quality reference genome.

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June 1, 2021  |  

Full-length transcript profiling with the Iso-Seq method for improved genome annotations

Incomplete annotation of genomes represents a major impediment to understanding biological processes, functional differences between species, and evolutionary mechanisms. Often, genes that are large, embedded within duplicated genomic regions, or associated with repeats are difficult to study by short-read expression profiling and assembly. In addition, most genes in eukaryotic organisms produce alternatively spliced isoforms, broadening the diversity of proteins encoded by the genome, which are difficult to resolve with short-read methods. Short-read RNA sequencing (RNA-seq) works by physically shearing transcript isoforms into smaller pieces and bioinformatically reassembling them, leaving opportunity for misassembly or incomplete capture of the full diversity of isoforms from genes of interest. In contrast, Single Molecule, Real-Time (SMRT) Sequencing directly sequences full-length transcripts without the need for assembly and imputation. Here we apply the Iso-Seq method (long-read RNA sequencing) to detect full-length isoforms and the new IsoPhase algorithm to retrieve allele-specific isoform information for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata).

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June 1, 2021  |  

A complete solution for full-length transcript sequencing using the PacBio Sequel II System

Long read mRNA sequencing methods such as PacBio’s Iso-Seq method offers high-throughput transcriptome profiling in prokaryotic and eukaryotic cells. By avoiding the transcript assembly problem and instead sequencing full-length cDNA, Iso-Seq has emerged as the most reliable technology for annotating isoforms and, in turn, improving proteome predictions in a wide variety of organisms. Improvements in library preparation, sequencing throughput, and bioinformatics has enabled the Iso-Seq method to be complete solution for transcript characterization. The Iso-Seq Express kit is a one-day library prep requiring 60-300 ng of total RNA. The PacBio Sequel II system produces 4-5 million full-length reads, sufficient to profile a whole human transcriptome. Finally, the SQANTI2 software is a powerful tool for categorizing the complex isoforms against reference annotations, while also incorporating orthogonal information such as CAGE peak data, public RNA-seq junction data, and ORF predictions.

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