June 1, 2021  |  

Comprehensive genome and transcriptome structural analysis of a breast cancer cell line using PacBio long read sequencing

Genomic instability is one of the hallmarks of cancer, leading to widespread copy number variations, chromosomal fusions, and other structural variations. The breast cancer cell line SK-BR-3 is an important model for HER2+ breast cancers, which are among the most aggressive forms of the disease and affect one in five cases. Through short read sequencing, copy number arrays, and other technologies, the genome of SK-BR-3 is known to be highly rearranged with many copy number variations, including an approximately twenty-fold amplification of the HER2 oncogene. However, these technologies cannot precisely characterize the nature and context of the identified genomic events and other important mutations may be missed altogether because of repeats, multi-mapping reads, and the failure to reliably anchor alignments to both sides of a variation. To address these challenges, we have sequenced SK-BR-3 using PacBio long read technology. Using the new P6-C4 chemistry, we generated more than 70X coverage of the genome with average read lengths of 9-13kb (max: 71kb). Using Lumpy for split-read alignment analysis, as well as our novel assembly-based algorithms for finding complex variants, we have developed a detailed map of structural variations in this cell line. Taking advantage of the newly identified breakpoints and combining these with copy number assignments, we have developed an algorithm to reconstruct the mutational history of this cancer genome. From this we have discovered a complex series of nested duplications and translocations between chr17 and chr8, two of the most frequent translocation partners in primary breast cancers, resulting in amplification of HER2. We have also carried out full-length transcriptome sequencing using PacBio’s Iso-Seq technology, which has revealed a number of previously unrecognized gene fusions and isoforms. Combining long-read genome and transcriptome sequencing technologies enables an in-depth analysis of how changes in the genome affect the transcriptome, including how gene fusions are created across multiple chromosomes. This analysis has established the most complete cancer reference genome available to date, and is already opening the door to applying long-read sequencing to patient samples with complex genome structures.


June 1, 2021  |  

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.


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.


April 21, 2020  |  

Full-Length Transcriptome Sequencing and the Discovery of New Transcripts in the Unfertilized Eggs of Zebrafish (Danio rerio).

Understanding early gene expression in zebrafish embryos is a prerequisite for developmental biology research. In this study, 1,629,447 polymerase reads were obtained from the unfertilized eggs of zebrafish via full-length transcriptome sequencing using the PacBio RS II platform first. Then, 102,920 unique isoforms were obtained by correction, clustering and comparison with the zebrafish genome. 12,782 genes in the genome were captured, accounting for 39.71% of the all annotated genes. Approximately 62.27% of the 12,782 genes have been alternatively spliced. GO and KEGG annotations revealed that the unfertilized eggs primarily stored genes that participate in RNA processing and nuclear protein complex composition. According to this PacBio data that aligned with the genome, 3,970 fusion genes, 819 ncRNAs, and 84 new transcripts were predicted. Illumina RNA-seq and RT-qPCR detection found that the expression of two new transcripts, PB.5289.1 and PB.10209.1, were significantly up-regulated at the 2-cell stage and down-regulated rapidly thereafter, suggesting their involvement in minor ZGA during early embryonic development. This study indicated that the unfertilized eggs of zebrafish may have retained genes directly related to cell division and development to initiate the subsequent development in a limited space and time. On the other hand, NTRs or new transcriptome regions in the genome were discovered, which provided new clues regarding ZGA of MZT during early embryonic development in fish.Copyright © 2019 Mehjabin et al.


April 21, 2020  |  

Full-length transcriptome sequences obtained by a combination of sequencing platforms applied to heat shock proteins and polyunsaturated fatty acids biosynthesis in Pyropia haitanensis

Pyropia haitanensis is a high-yield commercial seaweed in China. Pyropia haitanensis farms often suffer from problems such as severe germplasm degeneration, while the mechanisms underlying resistance to abiotic stresses remain unknown because of lacking genomic information. Although many previous studies focused on using next-generation sequencing (NGS) technologies, the short-read sequences generated by NGS generally prevent the assembly of full-length transcripts, and then limit screening functional genes. In the present study, which was based on hybrid sequencing (NGS and single-molecular real-time sequencing) of the P. haitanensis thallus transcriptome, we obtained high-quality full-length transcripts with a mean length of 2998 bp and an N50 value of 3366 bp. A total of 14,773 unigenes (93.52%) were annotated in at least one database, while approximately 60% of all unigenes were assembled by short Illumina reads. Moreover, we herein suggested that the genes involved in the biosynthesis of polyunsaturated fatty acids and heat shock proteins play an important role in the process of development and resistance to abiotic stresses in P. haitanensis. The present study, together with previously published ones, may facilitate seaweed transcriptome research.


April 21, 2020  |  

Identification of putative genes for polyphenol biosynthesis in olive fruits and leaves using full-length transcriptome sequencing.

Olive (Olea europaea) is a rich source of valuable bioactive polyphenols, which has attracted widespread interest. In this study, we combined targeted metabolome, Pacbio ISOseq transcriptome, and Illumina RNA-seq transcriptome to investigate the association between polyphenols and gene expression in the developing olive fruits and leaves. A total of 12 main polyphenols were measured, and 122 transcripts of 17 gene families, 101 transcripts of 9 gene families, and 106 transcripts of 6 gene families that encode for enzymes involved in flavonoid, oleuropein, and hydroxytyrosol biosynthesis were separately identified. Additionally, 232 alternative splicing events of 18 genes related to polyphenol synthesis were analyzed. This is the first time that the third generations of full-length transcriptome technology were used to study the gene expression pattern of olive fruits and leaves. The results of transcriptome combined with targeted metabolome can help us better understand the polyphenol biosynthesis pathways in the olive.Copyright © 2019 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

A global survey of full-length transcriptome of Ginkgo biloba reveals transcript variants involved in flavonoid biosynthesis

Ginkgo biloba, which contains flavonoids as bioactive components, is widely used in traditional Chinese medicine. Increasing the flavonoid production of medicinal plants through genetic engineering generally focuses on the key genes involved in flavonoid biosynthesis. However, the molecular mechanisms underlying such biosynthesis are not yet well understood. To understand these mechanisms, a combination of second-generation sequencing (SGS) and single-molecule real-time (SMRT) sequencing was applied to G. biloba. Eight tissues were sampled for SMRT sequencing to generate a high-quality, full-length transcriptome database. From 23.36 Gb clean reads, 12,954 alternative polyadenylation events, 12,290 alternative splicing events, 929 fusion transcripts, 2,286 novel transcripts, and 1,270 lncRNAs were predicted by removing redundant reads. Further studies reveal that 7 AS, 5 lncRNA, and 6 fusion gene events were identified in flavonoid biosynthesis. A total of 12 gene modules were revealed to be involved in flavonoid metabolism structural genes and transcription factors by constructing co-expression networks. Weighted gene coexpression network analysis (WGCNA) analysis reveals that some hub genes operate during the biosynthesis by identifying transcription factors (TFs) and structure genes. Seven key hub genes were also identified by analyzing the correlation between gene expression level and flavonoids content. The results highlight the importance of SMRT sequencing of the full-length transcriptome in improving genome annotation and elucidating the gene regulation of flavonoid biosynthesis in G. biloba by providing a comprehensive set of reference transcripts.


April 21, 2020  |  

Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.


April 21, 2020  |  

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.


April 21, 2020  |  

Comparative transcriptome analyses of genes involved in sulforaphane metabolism at different treatment in Chinese kale using full-length transcriptome sequencing.

Sulforaphane is a natural isothiocyanate available from cruciferous vegetables with multiple characteristics including antioxidant, antitumor and anti-inflammatory effect. Single-molecule real-time (SMRT) sequencing has been used for long-read de novo assembly of plant genome. Here, we investigated the molecular mechanism related to glucosinolates biosynthesis in Chinese kale using combined NGS and SMRT sequencing.SMRT sequencing produced 185,134 unigenes, higher than 129,325 in next-generation sequencing (NGS). NaCl (75?mM), methyl jasmonate (MeJA, 40?µM), selenate (Se, sodium selenite 100?µM), and brassinolide (BR, 1.5?µM) treatment induced 6893, 13,287, 13,659 and 11,041 differentially expressed genes (DEGs) in Chinese kale seedlings comparing with control. These genes were associated with pathways of glucosinolates biosynthesis, including phenylalanine, tyrosine and tryptophan biosynthesis, cysteine and methionine metabolism, and glucosinolate biosynthesis. We found NaCl decreased sulforaphane and glucosinolates (indolic and aliphatic) contents and downregulated expression of cytochrome P45083b1 (CYP83b1), S-alkyl-thiohydroximatelyase or carbon-sulfur lyase (SUR1) and UDP-glycosyltransferase 74B1 (UGT74b1). MeJA increased sulforaphane and glucosinolates contents and upregulated the expression of CYP83b1, SUR1 and UGT74b1; Se increased sulforaphane; BR increased expression of CYP83b1, SUR1 and UGT74b1, and increased glucosinolates contents. The desulfoglucosinolate sulfotransferases ST5a_b_c were decreased by all treatments.We confirmed that NaCl inhibited the biosynthesis of both indolic and aliphatic glucosinolates, while MeJA and BR increased them. MeJA and BR treatments, conferred the biosynthesis of glucosinolates, and Se and MeJA contributed to sulforaphane in Chinese kale via regulating the expression of CYP83b1, SUR1 and UGT74b1.


April 21, 2020  |  

Full-length transcriptome sequencing and methyl jasmonate-induced expression profile analysis of genes related to patchoulol biosynthesis and regulation in Pogostemon cablin.

Pogostemon cablin (Blanco) Benth. (Patchouli) is an important aromatic and medicinal plant and widely used in traditional Chinese medicine as well as in the perfume industry. Patchoulol is the primary bioactive component in P. cablin, its biosynthesis has attracted widespread interests. Previous studies have surveyed the putative genes involved in patchoulol biosynthesis using next-generation sequencing method; however, technical limitations generated by short-read sequencing restrict the yield of full-length genes. Additionally, little is known about the expression pattern of genes especially patchoulol biosynthesis related genes in response to methyl jasmonate (MeJA). Our understanding of patchoulol biosynthetic pathway still remained largely incomplete to date.In this study, we analyzed the morphological character and volatile chemical compounds of P. cablin cv. ‘Zhanxiang’, and 39 volatile chemical components were detected in the patchouli leaf using GC-MS, most of which were sesquiterpenes. Furthermore, high-quality RNA isolated from leaves and stems of P. cablin were used to generate the first full-length transcriptome of P. cablin using PacBio isoform sequencing (Iso-Seq). In total, 9.7 Gb clean data and 82,335 full-length UniTransModels were captured. 102 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis. Accorded with the uptrend of patchoulol content, the vast majority of genes related to the patchoulol biosynthesis were up-regulated after MeJA treatment, indicating that MeJA led to an increasing synthesis of patchoulol through activating the expression level of genes involved in biosynthesis pathway of patchoulol. Moreover, expression pattern analysis also revealed that transcription factors participated in JA regulation of patchoulol biosynthesis were differentially expressed.The current study comprehensively reported the morphological specificity, volatile chemical compositions and transcriptome characterization of the Chinese-cultivated P. cablin cv. ‘Zhanxiang’, these results contribute to our better understanding of the physiological and molecular features of patchouli, especially the molecular mechanism of biosynthesis of patchoulol. Our full-length transcriptome data also provides a valuable genetic resource for further studies in patchouli.


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