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Auto Tag: Isoform sequencing

June 1, 2021  |  

SMRT Sequencing of DNA and RNA samples extracted from formalin-fixed and paraffin-embedded tissues

Recent advances in next-generation sequencing have led to the increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule, Real-Time (SMRT) Sequencing offers a unique advantage in that it allows direct analysis of FFPE samples without amplification. However, obtaining ample long-read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. DNA samples extracted from FFPE often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and amplification. Additionally, the quality and quantity of the recovered DNA also vary depending on the extraction methods used. We have evaluated the Adaptive Focused Acoustics (AFA™) system by Covaris as a method for obtaining high molecular weight DNA suitable for SMRTbell template preparation and subsequent single molecule sequencing. Using this method, genomic DNA was extracted from normal kidney FFPE scrolls acquired from Cooperative Human Tissue Network (CHTN), University of Pennsylvania. Damaged sites present in the extracted DNA were repaired using a DNA Damage Repair step, and the treated DNA was constructed into SMRTbell libraries suitable for sequencing on the PacBio RS II System. Using the same repaired DNA, we also tested PCR efficiency of target gene regions of up to 5 kb. The resulting amplicons were constructed into SMRTbell templates for full-length sequencing on the PacBio RS II System. We found the Adaptive Focused Acoustics (AFA) system combined with truXTRAC™ by Covaris to be effective and efficient. This system is easy and simple to use, and the resulting DNA is compatible with SMRTbell library preparation for targeted and whole genome SMRT Sequencing. The data presented here demonstrates single molecule sequencing of DNA samples extracted from tissues embedded in FFPE.

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June 1, 2021  |  

Candidate gene screening using long-read sequencing

We have developed several candidate gene screening applications for both Neuromuscular and Neurological disorders. The power behind these applications comes from the use of long-read sequencing. It allows us to access previously unresolvable and even unsequencable genomic regions. SMRT Sequencing offers uniform coverage, a lack of sequence context bias, and very high accuracy. In addition, it is also possible to directly detect epigenetic signatures and characterize full-length gene transcripts through assembly-free isoform sequencing. In addition to calling the bases, SMRT Sequencing uses the kinetic information from each nucleotide to distinguish between modified and native bases.

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June 1, 2021  |  

Application-specific barcoding strategies for SMRT Sequencing

The increased sequencing throughput creates a need for multiplexing for several applications. We are here detailing different barcoding strategies for microbial sequencing, targeted sequencing, Iso-Seq full-length isoform sequencing, and Roche NimbleGen’s target enrichment method.

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June 1, 2021  |  

A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes such as APP, PSEN1 and PSEN2 that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form of late-onset AD. However, the identified SNPs are typically not the actual risk variants, but are in linkage disequilibrium with the presumed causative variant (Van Cauwenberghe C, et al., The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med 2015;18:421-430). Long-read sequencing together with hybrid-capture targeting technologies provides a powerful combination to target candidate genes/transcripts of interest. Shearing the genomic DNA to ~5 kb fragments and then capturing with probes that span the whole gene(s) of interest can provide uniform coverage across the entire region, identifying variants and allowing for phasing into two haplotypes. Furthermore, capturing full-length cDNA from the same sample using the same capture probes can also provide an understanding of isoforms that are generated and allow them to be assigned to their corresponding haplotype. Here we present a method for capturing genomic DNA and cDNA from an AD sample using a panel of probes targeting approximately 20 late-onset AD candidate genes which includes CLU, ABCA7, CD33, TREM2, TOMM40, PSEN2, APH1 and BIN1. By combining xGen® Lockdown® probes with SMRT Sequencing, we provide completely sequenced candidate genes as well as their corresponding transcripts. In addition, we are also able to evaluate structural variants that due to their size, repetitive nature, or low sequence complexity have been un-sequenceable using short-read technologies.

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June 1, 2021  |  

Alternative splicing in FMR1 premutations carriers

Over 40% of males and ~16% of female carriers of a FMR1 premutation allele (55-200 CGG repeats) are at risk for developing Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), an adult onset neurodegenerative disorder while, about 20% of female carriers will develop Fragile X-associated Primary Ovarian Insufficiency (FXPOI), in addition to a number of adult-onset clinical problems (FMR1 associated disorders). Marked elevation in FMR1 mRNA levels have been observed with premutation alleles and the resulting RNA toxicity is believed to be the leading molecular mechanism proposed for these disorders. The FMR1 gene, as many housekeeping genes, undergoes alternative splicing. Using long-read isoform sequencing (SMRT) and qRT-PCR we have recently reported that, although the relative abundance of all FMR1 mRNA isoforms is significantly increased in the premutation group compared to controls, there is a disproportionate increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14. In total, we confirmed the existence of 16 out of 24 predicted isoforms in our samples. However, it is unknown, which isoforms, when overexpressed, may contribute to the premutation pathology. To address this question we have further defined the transcriptional FMR1 isoforms distribution pattern in different tissues, including heart, muscle, brain and testis derived from FXTAS premutation carriers and age-matched controls. Preliminary data indicates the presence of a transcriptional signature of the FMR1 gene, which clusters more by individual than by tissue type. We identified additional isoforms than the 16 reported in our previous study, including a group with particular splice patterns that were observed only in premutations but not in controls. Our findings suggest that the characterization of expression levels of the different FMR1 isoforms is fundamental for understanding the regulation of the FMR1 gene as well as for elucidating the mechanism(s) by which “toxic gain of function” of the FMR1 mRNA may play a role in FXTAS and/or in the other FMR1-associated conditions. In addition to the elevated levels of FMR1 isoforms, the altered abundance/ratio of the corresponding FMRP isomers may affect the overall function of FMRP in premutations.

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June 1, 2021  |  

Full-length cDNA sequencing on the PacBio Sequel platform

The protein coding potential of most plant and animal genomes is dramatically increased via alternative splicing. Identification and annotation of expressed mRNA isoforms is critical to the understanding of these complex organisms. 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. The PacBio Sequel platform has improved throughput thereby increasing the number of full-length transcripts per SMRT Cell. Furthermore, loading enhancements on the Sequel instrument have decreased the need for size fractionation steps. We have optimized the Iso-Seq library preparation process for use on the Sequel platform. Here, we demonstrate the capabilities of the Iso-Seq method on the Sequel system using cDNAs from the maize (Zea mays) inbred line B73. Full-length cDNA from six diverse tissues were barcoded, pooled, and sequenced on the PacBio Sequel system using a combination of size-selected and non-size-selected SMRTbell libraries. The results highlight the value of full-length transcripts for genome annotations and analysis of alternative splicing.

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June 1, 2021  |  

Using the PacBio IsoSeq method to search for novel colorectal cancer biomarkers

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.

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June 1, 2021  |  

A method for the identification of variants in Alzheimer’s disease candidate genes and transcripts using hybridization capture combined with long-read sequencing

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes such as APP, PSEN1 and PSEN2 that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form of late-onset AD. However, the identified SNPs are typically not the actual causal variants, but are in linkage disequilibrium with the presumed causative variant (Van Cauwenberghe C, et al., The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med 2015;18:421-430).

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June 1, 2021  |  

SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants

Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being developed as a predictive biomarker of primary resistance to androgen receptor (AR)-targeted therapies in CRPC. Multiple additional AR-V species are expressed in clinical CRPC, but the extent to which these may be co-expressed with AR-V7 or predict resistance is not known.

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June 1, 2021  |  

Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel platform

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 – 10 kb, fully intact RNA molecules can be sequenced using SMRT® 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.

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June 1, 2021  |  

Screening for causative structural variants in neurological disorders using long-read sequencing

Over the past decades neurological disorders have been extensively studied producing a large number of candidate genomic regions and candidate genes. The SNPs identified in these studies rarely represent the true disease-related functional variants. However, more recently a shift in focus from SNPs to larger structural variants has yielded breakthroughs in our understanding of neurological disorders.Here we have developed candidate gene screening methods that combine enrichment of long DNA fragments with long-read sequencing that is optimized for structural variation discovery. We have also developed a novel, amplification-free enrichment technique using the CRISPR/Cas9 system to target genomic regions.We sequenced gDNA and full-length cDNA extracted from the temporal lobe for two Alzheimer’s patients for 35 GWAS candidate genes. The multi-kilobase long reads allowed for phasing across the genes and detection of a broad range of genomic variants including SNPs to multi-kilobase insertions, deletions and inversions. In the full-length cDNA data we detected differential allelic isoform complexity, novel exons as well as transcript isoforms. By combining the gDNA data with full-length isoform characterization allows to build a more comprehensive view of the underlying biological disease mechanisms in Alzheimer’s disease. Using the novel PCR-free CRISPR-Cas9 enrichment method we screened several genes including the hexanucleotide repeat expansion C9ORF72 that is associated with 40% of familiar ALS cases. This method excludes any PCR bias or errors from an otherwise hard to amplify region as well as preserves the basemodication in a single molecule fashion which allows you to capture mosaicism present in the sample.

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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  |  

De novo assembly and preliminary annotation of the Schizocardium californicum genome

Animals in the phylum Hemichordata have provided key understanding of the origins and development of body patterning and nervous system organization. However, efforts to sequence and assemble the genomes of highly heterozygous non-model organisms have proven to be difficult with traditional short read approaches. Long repetitive DNA structures, extensive structural variation between haplotypes in polyploid species, and large genome sizes are limiting factors to achieving highly contiguous genome assemblies. Here we present the highly contiguous de novo assembly and preliminary annotation of an indirect developing hemichordate genome, Schizocardium californicum, using SMRT Sequening long reads.

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June 1, 2021  |  

Haplotyping of full-length transcript reads from long-read sequencing can reveal allelic imbalances in isoform expression

The Pacific Biosciences Iso-Seq method, which can produce high-quality isoform sequences of 10 kb and longer, has been used to annotate many important plant and animal genomes. Here, we develop an algorithm called IsoPhase that postprocesses Iso-Seq data to retrieve allele specific isoform information. Using simulated data, we show that for both diploid and tetraploid genomes, IsoPhase results in good SNP recovery with low FDR at error rates consistent with CCS reads. We apply IsoPhase to a haplotyperesolved genome assembly and multiple fetal tissue Iso-Seq dataset from a F1 cross of Angus x Brahman cattle subspecies. IsoPhase-called haplotypes were validated by the phased assembly and demonstrate the potential for revealing allelic imbalances in isoform expression.

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June 1, 2021  |  

Characterizing the pan-genome of maize with PacBio SMRT Sequencing

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.

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