Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test that detects the blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection.
Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. 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. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences needed to discover biomarkers for early detection and cancer stratification, to fully characterize gene fusion events, and to elucidate drug resistance mechanisms. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 â€“ 10 kb, fully intact RNA molecules can be sequenced using SMRTÂ® Sequencing without requiring fragmentation or post-sequencing assembly. However, some cancer research applications have presented a challenge for the Iso-Seq protocol, due to the combination of limited sample input and the need to deeply sequence heterogenous samples. Here we report the optimization of the Iso-Seq library preparation protocol for the PacBio Sequel platform and its application to cancer cell lines and tumor samples. We demonstrate how loading enhancements on the higher-throughput Sequel instrument have decreased the need for size fractionation steps, reducing sample input requirements while simultaneously simplifying the sample preparation workflow and increasing the number of full-length transcripts per SMRT Cell.
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.
Maize is an amazingly diverse crop. A study in 20051 demonstrated that half of the genome sequence and one-third of the gene content between two inbred lines of maize were not shared. This diversity, which is more than two orders of magnitude larger than the diversity found between humans and chimpanzees, highlights the inability of a single reference genome to represent the full pan-genome of maize and all its variants. Here we present and review several efforts to characterize the complete diversity within maize using the highly accurate long reads of PacBio Single Molecule, Real-Time (SMRT) Sequencing. These methods provide a framework for a pan-genomic approach that can be applied to studies of a wide variety of important crop species.
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.
Allelic specificity of immunoglobulin heavy chain ([email protected]) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing
Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).
An important need in analyzing complex genomes is the ability to separate and phase haplotypes. While whole genome assembly can deliver this information, it cannot reveal whether there is allele-specific gene or isoform expression. The PacBio Iso-Seq method, which can produce high-quality transcript sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. We present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents and two F1 cross hybrids. We validated the majority of the SNPs called with IsoPhase against matching short read data and identified cases of allele-specific, gene-level and isoform-level expression.
Library prep and bioinformatics improvements for full-length transcript sequencing on the PacBio Sequel System
The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. Here we describe an improved, simplified library workflow and analysis pipeline that reduces library preparation time, RNA input, and cost. The Iso-Seq V2 Express workflow is a one day protocol that requires only ~300 ng of total RNA input while also reducing the number of reverse transcription and amplification steps down to single reactions. Compared with the previous workflow, the Iso-Seq V2 Express workflow increases the percentage of full-length (FL) reads while achieving a higher average transcript length. At the same time, the Iso-Seq 3 analysis recently released in the SMRT Link 6.0 software is a major improvement over previous versions. Iso-Seq 3 is highly accurate at detecting and removing library artifacts (TSO and RT artifacts) as well as differentiating barcodes on multiplexed samples. Iso-Seq 3 achieves the same output performance in high-quality transcript sequences compared to previous versions while reducing the runtime and memory usage dramatically.
Full-length transcriptome sequencing of melanoma cell line complements long-read assessment of genomic rearrangements
Transcriptome sequencing has proven to be an important tool for understanding the biological changes in cancer genomes including the consequences of structural rearrangements. Short read sequencing has been the method of choice, as the high throughput at low cost allows for transcript quantitation and the detection of even rare transcripts. However, the reads are generally too short to reconstruct complete isoforms. Conversely, long-read approaches can provide unambiguous full-length isoforms, but lower throughput has complicated quantitation and high RNA input requirements has made working with cancer samples challenging. Recently, the COLO 829 cell line was sequenced to 50-fold coverage with PacBio SMRT Sequencing. To validate and extend the findings from this effort, we have generated long-read transcriptome data using an updated PacBio Iso-Seq method, the results of which will be shared at the AACR 2019 General Meeting. With this complimentary transcriptome data, we demonstrate how recent innovations in the PacBio Iso-Seq method sample preparation and sequencing chemistry have made long-read sequencing of cancer transcriptomes more practical. In particular, library preparation has been simplified and throughput has increased. The improved protocol has reduced sample prep time from several days to one day while reducing the sample input requirements ten-fold. In addition, the incorporation of unique molecular identifier (UMI) tags into the workflow has improved the bioinformatics analysis. Yield has also increased, with v3 sequencing chemistry typically delivering > 30 Gb per SMRT Cell 1M. By integrating long and short read data, we demonstrate that the Iso-Seq method is a practical tool for annotating cancer genomes with high-quality transcript information.
The PacBio Iso-Seq method produces high-quality, full-length transcripts and can characterize a whole transcriptome with a single SMRT Cell 8M. We sequenced an Alzheimer whole brain sample on a single SMRT Cell 8M on the Sequel II System. Using the Iso-Seq bioinformatics pipeline followed by SQANTI2 analysis, we detected 162,290 transcripts for 17,670 genes up to 14 kb in length. More than 60% of the transcripts are novel isoforms, the vast majority of which have supporting cage peak data and polyadenylation signals, demonstrating the utility of long-read sequencing for human disease research.
The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more transcripts. Most importantly, the method is supported by a maturing suite of official and community-developed tools. The SMRT Link Iso-Seq application outputs high-quality (>99% accurate), full-length transcript sequences that can optionally be mapped to a reference genome for a single SMRT Cell worth of data in 6-9 hours. For example, the SQANTI2 tool classifies Iso-Seq transcripts against a reference annotation, filters potential library artifacts, and processes information from both long read-only and short read-based quantification. IsoPhase is a tool for identifying allele-specific isoform expression. Cogent has been used to process Iso-Seq transcripts in a genome-independent manner to assess genome assemblies. Finally, IsoAnnot is an up-and-coming tool for identifying differential isoform expression across different samples. We describe how these tools complement each other and provide guidelines to make the best use out of Iso-Seq data for understanding transcriptomes.