HiFi Sequencing of Full-length RNA to Characterize Transcriptomes

HiFi Sequencing of Full-length RNA to Characterize Transcriptomes

This deck provides information on highly accurate, long read sequencing of full-length RNA to characterize transcriptomes and several example use cases.

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Michelle Vierra

July 16, 2020
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  1. PacBio HiFi Sequencing Allows You to Explore the Transcriptomes of

    Plants and Animals with Confidence Learn How HiFi Sequencing of Full-Length RNA Can Help You Advance Your Research
  2. WHAT ARE HIFI READS? www.pacb.com/HiFi -They are long -Tens of

    kilobases -They are highly accurate -Long reads with ≥Q20 (99%) single-molecule accuracy -They have single-molecule resolution -Sequence DNA or cDNA -They are unbiased -No DNA amplification, least GC content and sequence complexity bias
  3. WHY DO HIFI READS MATTER FOR RNA SEQUENCING? Short Reads

    HiFi Reads Few reads spanning junctions Additional inference required to parse isoforms Full-length transcripts capture all splice junctions Gene with Several Transcript Isoforms Reads span all junctions
  4. WHAT CAN YOU DO WITH FULL-LENGTH RNA SEQUENCING? - Accurately

    characterize transcript diversity - Gain poly-adenylation site information - Profile expression at isoform resolution - Catalog allele-specific expression differences - Annotate sex chromosomes - Identify functional implications of alternative splicing Learn More about Each of These and the Benefits HiFi Reads Provide
  5. ACCURATELY CHARACTERIZE TRANSCRIPT DIVERSITY “Reliable identification of FL transcripts is

    clearly an advantage of the PacBio data, which has enabled us to infer considerably more alternatively spliced transcripts than before.” Zuo, C. et al. (2018) Revealing the transcriptomic complexity of switchgrass by PacBio long-read sequencing. BMC Biotechnology for Biofuels. 11:170 Previous gene model PacBio transcripts
  6. GAIN POLY-ADENYLATION SITE INFORMATION Wang, B. et al. (2018) A

    comparative transcriptional landscape of maize and sorghum obtained by single-molecule sequencing. Genome Research 28:1-12 “We generated comprehensive and high-resolution maps of genome-wide poly(A) sites, allowing systematic characterization of the role of APA in… agronomically important species.” Alternative cleavage sites Top 10 alternative polyadenylation (APA) motifs for maize (left) and sorghum (right)
  7. PROFILE EXPRESSION AT ISOFORM RESOLUTION Minio, A. et al. (2018)

    Isoform-scale annotation and expression profiling of the Cabernet Sauvignon transcriptome using single-molecule sequencing of full- length cDNA. bioRxiv 65 of the 500+ Cabernet Sauvignon-specific transcripts were found to be differentially regulated during ripening
  8. CATALOG ALLELE-SPECIFIC EXPRESSION DIFFERENCES Wang, B. et al. (2019) Variant

    Phasing and Haplotypic Expression from Single-molecule Long-read Sequencing in Maize. bioRxiv SNPs are inherited with Ki11 in only one version of the hybrid SNPs are inherited at 50/50 ratio despite the type of hybrid
  9. ANNOTATE SEX CHROMOSOMES McKernan, K. et al. (2020) Sequence and

    annotation of 42 cannabis genomes reveals extensive copy number variation in cannabinoid synthesis and pathogen resistance genes. bioRxiv. “We were able to confirm putative Y contigs and male-specific genes.”
  10. IDENTIFY FUNCTIONAL IMPLICATIONS OF ALTERNATIVE SPLICING Shawn Trojahn, PacBio User

    Group Meeting Presentation: Using Grizzly Bears to Unlock the Biomedical Promise of Hibernation “Now we’re able to see that with 86 of these genes one isoform is up and one isoform is down, meaning possibly these isoforms are performing different functions between the seasons. This is something we did not pick up on previously because we just used gene-level analysis.”
  11. FULL-LENGTH RNA SEQUENCING WITH PACBIO SYSTEMS IS AFFORDABLE A single

    SMRT Cell 8M is all you need! *Study design, sample type, and level of multiplexing may affect the number of SMRT Cells 8M required. †All prices are listed in USD and cost may vary by region. Pricing includes library and sequencing reagents run on your Sequel II System and does not include instrument amortization or other reagents. SMRT Sequencing Applications Number of SMRT Cells 8M* PacBio Consumable Estimated Costs (US List Price)† Whole Transcriptome Characterize alternative splicing with full- length transcripts 1 $1300/sample Genome Annotation Sequence full-length transcripts and multiplex up to 8 tissues 1 $185/tissue
  12. -For more information on the PacBio RNA sequencing and how

    it applies to your research, please visit www.pacb.com/agbio-rna GET STARTED WITH PACBIO FULL-LENGTH RNA SEQUENCING -To get started with a free project consultation with a PacBio scientist, please visit www.pacb.com/scientist
  13. For Research Use Only. Not for use in diagnostic procedures.

    © Copyright 2020 by Pacific Biosciences of California, Inc. All rights reserved. Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq, and Sequel are trademarks of Pacific Biosciences. FEMTO Pulse and Fragment Analyzer are trademarks of Agilent Technologies Inc. All other trademarks are the sole property of their respective owners. www.pacb.com