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Auto Tag: PolyA tail

June 1, 2021  |  

Isoform sequencing: Unveiling the complex landscape of the eukaryotic transcriptome on the PacBio RS II.

Alternative splicing of RNA is an important mechanism that increases protein diversity and is pervasive in the most complex biological functions. While advances in RNA sequencing methods have accelerated our understanding of the transcriptome, isoform discovery remains computationally challenging due to short read lengths. Here, we describe the Isoform Sequencing (Iso-Seq) method using long reads generated by the PacBio RS II. We sequenced rat heart and lung RNA using the Clontech® SMARTer® cDNA preparation kit followed by size selection using agarose gel. Additionally, we tested the BluePippin™ device from Sage Science for efficiently extracting longer transcripts = 3 kb. Post-sequencing, we developed a novel isoform-level clustering algorithm to generate high-quality transcript consensus sequences. We show that our method recovered alternative splice forms as well as alternative stop sites, antisense transcription, and retained introns. To conclude, the Iso-Seq method provides a new opportunity for researchers to study the complex eukaryotic transcriptome even in the absence of reference genomes or annotated transcripts.

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June 1, 2021  |  

A novel analytical pipeline for de novo haplotype phasing and amplicon analysis using SMRT Sequencing technology.

While the identification of individual SNPs has been readily available for some time, the ability to accurately phase SNPs and structural variation across a haplotype has been a challenge. With individual reads of an average length of 9 kb (P5-C3), and individual reads beyond 30 kb in length, SMRT Sequencing technology allows the identification of mutation combinations such as microdeletions, insertions, and substitutions without any predetermined reference sequence. Long- amplicon analysis is a novel protocol that identifies and reports the abundance of differing clusters of sequencing reads within a single library. Graphs generated via hierarchical clustering of individual sequencing reads are used to generate Markov models representing the consensus sequence of individual clusters found to be significantly different. Long-amplicon analysis is capable of differentiating between underlying sequences that are 99.9% similar, which is suitable for haplotyping and differentiating pseudogenes from coding transcripts. This protocol allows for the identification of structural variation in the MUC5AC gene sequence, despite the presence of a gap in the current genome assembly, and can also be used for HLA haplotyping. Clustering can also been applied to identify full length transcripts for the purpose of estimating consensus sequences and enumerating isoform types. Long-amplicon analysis allows for the elucidation of complex regions otherwise missed by other sequencing technologies, which may contribute to the diagnosis and understanding of otherwise complex diseases.

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June 1, 2021  |  

Isoform sequencing: Unveiling the complex landscape in eukaryotic transcriptome on the PacBio RS II.

Advances in RNA sequencing have accelerated our understanding of the transcriptome, however isoform discovery remains challenging due to short read lengths. The Iso-Seq Application provides a new alternative to sequence full-length cDNA libraries using long reads from the PacBio RS II. Identification of long and often rare isoforms is demonstrated with rat heart and lung RNA prepared using the Clontech® SMARTer® cDNA preparation kit, followed by agarose-gel size selection in fractions of 1-2 kb, 2-3 kb and 3-6 kb. For each tissue, 1.8 and 1.2 million reads were obtained from 32 and 26 SMRT Cells, respectively. Filtering for reads with both adapters and polyA tail signals yielded >50% putative full-length transcripts. To improve consensus accuracy, we developed an isoform-level clustering algorithm ICE (Iterative Clustering for Error Correction), and polished full-length consensus sequences from ICE using Quiver. This method generated full-length transcripts up to 4.5 kb with = 99% post-correction accuracy. Compared with known rat genes, the Iso-Seq method not only recovered the majority of currently annotated isoforms, but also several unannotated novel isoforms with identified homologs in the RefSeq database. Additionally, alternative stop sites, extended UTRs, and retained introns were detected.

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June 1, 2021  |  

Single Molecule, Real-Time sequencing of full-length cDNA transcripts uncovers novel alternatively spliced isoforms.

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 such as structure, function, or subcellular localization. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be underscored. 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 (avg. read length: 10-15 kb) 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. The standard Iso-Seq protocol workflow available for all researchers is presented using a deep dataset of full- length cDNA sequences from the MCF-7 cancer cell line, and multiple tissues (brain, heart, and liver). Detected novel transcripts approaching 10 kb and alternative splicing events are highlighted. Even in extensively profiled samples, the method uncovered large numbers of novel alternatively spliced isoforms and previously unannotated genes.

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June 1, 2021  |  

Full-length isoform sequencing of the human MCF-7 cell line using PacBio long reads.

While advances in RNA sequencing methods have accelerated our understanding of the human transcriptome, isoform discovery remains a challenge because short read lengths require complicated assembly algorithms to infer the contiguity of full-length transcripts. With PacBio’s long reads, one can now sequence full-length transcript isoforms up to 10 kb. The PacBio Iso- Seq protocol produces reads that originate from independent observations of single molecules, meaning no assembly is needed. Here, we sequenced the transcriptome of the human MCF-7 breast cancer cell line using the Clontech SMARTer® cDNA preparation kit and the PacBio RS II. Using PacBio Iso-Seq bioinformatics software, we obtained 55,770 unique, full-length, high-quality transcript sequences that were subsequently mapped back to the human genome with = 99% accuracy. In addition, we identified both known and novel fusion transcripts. To assess our results, we compared the predicted ORFs from the PacBio data against a published mass spectrometry dataset from the same cell line. 84% of the proteins identified with the Uniprot protein database were recovered by the PacBio predictions. Notably, 251 peptides solely matched to the PacBio generated ORFs and were entirely novel, including abundant cases of single amino acid polymorphisms, cassette exon splicing and potential alternative protein coding frames.

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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  |  

From RNA to full-length transcripts: The PacBio Iso-Seq method for transcriptome analysis and genome annotation

A single gene may encode a surprising number of proteins, each with a distinct biological function. This is especially true in complex eukaryotes. 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. The PacBio Isoform Sequencing (Iso-Seq™) method employs long reads to sequence transcript isoforms from the 5’ end to their poly-A tails, eliminating the need for transcript reconstruction and inference. These long reads result in complete, unambiguous information about alternatively spliced exons, transcriptional start sites, and poly- adenylation sites. This allows for the characterization of the full complement of isoforms within targeted genes, or across an entire transcriptome. Here we present improved genome annotations for two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata), using the Iso-Seq method. We present graphical user interface and command line analysis workflows for the data sets. From brain total RNA, we characterize more than 15,000 isoforms in each species, 9% and 5% of which were previously unannotated in hummingbird and zebra finch, respectively. We highlight one example where capturing full-length transcripts identifies additional exons and UTRs.

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June 1, 2021  |  

Scalability and reliability improvements to the Iso-Seq analysis pipeline enables higher throughput sequencing of full-length cancer transcripts

The characterization of gene expression profiles via transcriptome sequencing has proven to be an important tool for characterizing how genomic rearrangements in cancer affect the biological pathways involved in cancer progression and treatment response. More recently, better resolution of transcript isoforms has shown that this additional level of information may be useful in stratifying patients into cancer subtypes with different outcomes and responses to treatment.1 The Iso-Seq protocol developed at PacBio is uniquely able to deliver full-length, high-quality cDNA sequences, allowing the unambiguous determination of splice variants, identifying potential biomarkers and yielding new insights into gene fusion events. Recent improvements to the Iso-Seq bioinformatics pipeline increases the speed and scalability of data analysis while boosting the reliability of isoform detection and cross-platform usability. Here we report evaluation of Sequel Iso-Seq runs of human UHRR samples with spiked-in synthetic RNA controls and show that the new pipeline is more CPU efficient and recovers more human and synthetic isoforms while reducing the number of false positives. We also share the results of sequencing the well-characterized HCC-1954 breast cancer and normal breast cell lines, which will be made publicly available. Combined with the recent simplification of the Iso-Seq sample preparation2, the new analysis pipeline completes a streamlined workflow for revealing the most comprehensive picture of transcriptomes at the throughput needed to characterize cancer samples.

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June 1, 2021  |  

Full-length cDNA sequencing of prokaryotic transcriptome and metatranscriptome samples

Next-generation sequencing has become a useful tool for studying transcriptomes. However, these methods typically rely on sequencing short fragments of cDNA, then attempting to assemble the pieces into full-length transcripts. Here, we describe a method that uses PacBio long reads to sequence full-length cDNAs from individual transcriptomes and metatranscriptome samples. We have adapted the PacBio Iso-Seq protocol for use with prokaryotic samples by incorporating RNA polyadenylation and rRNA-depletion steps. In conjunction with SMRT Sequencing, which has average readlengths of 10-15 kb, we are able to sequence entire transcripts, including polycistronic RNAs, in a single read. Here, we show full-length bacterial transcriptomes with the ability to visualize transcription of operons. In the area of metatranscriptomics, long reads reveal unambiguous gene sequences without the need for post-sequencing transcript assembly. We also show full-length bacterial transcripts sequenced after being treated with NEB’s Cappable-Seq, which is an alternative method for depleting rRNA and enriching for full-length transcripts with intact 5’ ends. Combining Cappable-Seq with PacBio long reads allows for the detection of transcription start sites, with the additional benefit of sequencing entire transcripts.

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June 1, 2021  |  

Single cell isoform sequencing (scIso-Seq) identifies novel full-length mRNAs and cell type-specific expression

Single cell RNA-seq (scRNA-seq) is an emerging field for characterizing cell heterogeneity in complex tissues. However, most scRNA-seq methodologies are limited to gene count information due to short read lengths. Here, we combine the microfluidics scRNA-seq technique, Drop-Seq, with PacBio Single Molecule, Real-Time (SMRT) Sequencing to generate full-length transcript isoforms that can be confidently assigned to individual cells. We generated single cell Iso-Seq (scIso-Seq) libraries for chimp and human cerebral organoid samples on the Dolomite Nadia platform and sequenced each library with two SMRT Cells 8M on the PacBio Sequel II System. We developed a bioinformatics pipeline to identify, classify, and filter full-length isoforms at the single-cell level. We show that scIso-Seq reveals full-length isoform information not accessible using short reads that can reveal differences between cell types and amongst different species.

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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.

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April 21, 2020  |  

Comprehensive transcriptome analysis reveals genes potentially involved in isoflavone biosynthesis in Pueraria thomsonii Benth.

Pueraria thomsonii Benth is an important medicinal plant. Transcriptome sequencing, unigene assembly, the annotation of transcripts and the study of gene expression profiles play vital roles in gene function research. However, the full-length transcriptome of P. thomsonii remains unknown. Here, we obtained 44,339 nonredundant transcripts of P. thomsonii by using the PacBio RS II Isoform and Illumina sequencing platforms, of which 43,195 were annotated genes. Compared with the expression levels in the plant roots, those of transcripts with a |fold change| = 4 and FDR < 0.01 in the leaves or stems were assigned as differentially expressed transcripts (DETs). In total, we found 9,225 DETs, 32 of which came from structural genes that were potentially involved in isoflavone biosynthesis. The expression profiles of 8 structural genes from the RNA-Seq data were validated by qRT-PCR. We identified 437 transcription factors (TFs) that were positively or negatively correlated with at least 1 of the structural genes involved in isoflavone biosynthesis using Pearson correlation coefficients (r) (r > 0.8 or r < -0.8). We also identified a total of 32 microRNAs (miRNAs), which targeted 805 transcripts. These miRNAs caused enriched function in 'ATP binding', 'defense response', 'ADP binding', and 'signal transduction'. Interestingly, MIR156a potentially promoted isoflavone biosynthesis by repressing SBP, and MIR319 promoted isoflavone biosynthesis by repressing TCP and HB-HD-ZIP. Finally, we identified 2,690 alternative splicing events, including that of the structural genes of trans-cinnamate 4-monooxygenase and pullulanase, which are potentially involved in the biosynthesis of isoflavone and starch, respectively, and of three TFs potentially involved in isoflavone biosynthesis. Together, these results provide us with comprehensive insight into the gene expression and regulation of P. thomsonii.

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April 21, 2020  |  

Hybrid sequencing-based personal full-length transcriptomic analysis implicates proteostatic stress in metastatic ovarian cancer.

Comprehensive molecular characterization of myriad somatic alterations and aberrant gene expressions at personal level is key to precision cancer therapy, yet limited by current short-read sequencing technology, individualized catalog of complete genomic and transcriptomic features is thus far elusive. Here, we integrated second- and third-generation sequencing platforms to generate a multidimensional dataset on a patient affected by metastatic epithelial ovarian cancer. Whole-genome and hybrid transcriptome dissection captured global genetic and transcriptional variants at previously unparalleled resolution. Particularly, single-molecule mRNA sequencing identified a vast array of unannotated transcripts, novel long noncoding RNAs and gene chimeras, permitting accurate determination of transcription start, splice, polyadenylation and fusion sites. Phylogenetic and enrichment inference of isoform-level measurements implicated early functional divergence and cytosolic proteostatic stress in shaping ovarian tumorigenesis. A complementary imaging-based high-throughput drug screen was performed and subsequently validated, which consistently pinpointed proteasome inhibitors as an effective therapeutic regime by inducing protein aggregates in ovarian cancer cells. Therefore, our study suggests that clinical application of the emerging long-read full-length analysis for improving molecular diagnostics is feasible and informative. An in-depth understanding of the tumor transcriptome complexity allowed by leveraging the hybrid sequencing approach lays the basis to reveal novel and valid therapeutic vulnerabilities in advanced ovarian malignancies.

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